Jolly PDF

S. Afr. J. Enol. Vitic., Vol. 27, No.
1, 2006
15
* Corresponding author: E-mail address: jollyn@arc.agric.za
** Present address: The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, Adelaide, SA 5064, Australia.
***The Fruit, Vine and Wine Institute of the Agricultural Research Council
The Role and Use of Non-Saccharomyces Yeasts in Wine Production
N.P. Jolly
1
*, O.P.H. Augustyn
1
and I.S. Pretorius
2
**
(1) ARC Infruitec-Nietvoorbij***, Private Bag X5026, 7599 Stellenbosch, South Africa.
(2) Institute for Wine Biotechnology, Department of Viticulture & Oenology, Stellenbosch University, Private Bag X1, 7602 Matieland
(Stellenbosch), South Africa.
Submitted for publication: September 2005
Accepted for publication: April 2006
Key words: Non-Saccharomyces, yeasts, vineyards, cellars, fermentation, wine.
The contribution by the numerous grape-must-associated non-Saccharomyces yeasts to wine fermentation has been
debated extensively. These yeasts, naturally present in all wine fermentations, are metabolically active and their
metabolites can impact on wine quality. Although often seen as a source of microbial spoilage, there is substantial
contrary evidence pointing to a positive contribution by these yeasts. The role of non-Saccharomyces yeasts in wine
fermentation is therefore receiving increasing attention by wine microbiologists in Old and New World wine
producing countries. Species that have been investigated for wine production thus far include those from the
Candida, Kloeckera, Hanseniaspora, Zygosaccharomyces, Schizosaccharomyces, Torulaspora, Brettanomyces,
Saccharomycodes, Pichia and Williopsis genera. In this review the use and role of non-Saccharomyces yeast in wine
production is presented and research trends are discussed.
INTRODUCTION
Wine is the product of a complex biological and biochemical
interaction between grapes (grape juice) and different microor-
ganisms (fungi, yeasts, lactic acid bacteria and acetic acid bacte-
ria) and the mycoviruses and bacteriophages affecting them
(Fleet, 2003). The process starts in the vineyard, continues
through fermentation and maturation, and concludes at packag-
ing. It is affected by the various viticultural and oenological prac-
tices available to the grape-grower and winemaker, respectively
(Regueiro et al., 1993). Of the microorganisms involved, it is the
yeasts that play the most important role; they conduct the alco-
holic fermentation (conversion of grape sugar to ethanol and
CO2). Furthermore, although wine flavour is directly determined
by grape variety, yeasts also affect wine flavour and quality by the
production and excretion of metabolites during growth and
through autolysis (Fleet, 1993, 2003; Lambrechts & Pretorius,
2000; Swiegers & Pretorius, 2005; Swiegers et al., 2005). In
some instances, yeasts can also act as spoilage organisms during
wine production (including maturation) and after packaging
(Loureiro & Malfeito-Ferreira, 2003). Yeasts present during fer-
mentation are derived from grapes and the vineyard, the equip-
ment used in the cellar, cellar surfaces and external sources such
as selected cultures that are added to facilitate the fermentation
process.
Since 1866, when Louis Pasteur first elucidated the bio-con-
version of grape juice into wine, this complex process and the
role of the yeast therein has been studied extensively. Yet, more
than 130 years later, there are many areas that are still not well
understood (Pretorius, 2000). This is especially the case for the
roles of the numerous non-Saccharomyces yeasts normally asso-
ciated with grape must and wine. These yeasts, naturally present
in all wine fermentations to a greater or lesser extent, are meta-
bolically active and their metabolites can impact on wine quality.
While they were originally seen as a source of microbial related
problems in wine production, winemakers, especially in Old
World countries, saw indigenous yeasts as integral to the authen-
ticity of their wines as these yeasts impart distinct regional and
other desirable characteristics (Amerine et al., 1972; Jackson,
1994). Evidence supporting this view has been published (Fleet,
1990; Heard, 1999) and the role of the non-Saccharomyces yeasts
in wine fermentation is receiving increasingly more attention by
wine microbiologists in both Old and New World wine-producing
countries.
YEAST CLASSIFICATION
Yeasts can be defined as unicellular fungi, either ascomycetous or
basidiomycetous, that have vegetative states which predominant-
ly reproduce by budding or fission and which do not form their
sexual states within or on a fruiting body (Kurtzman & Fell,
1998a).
Current taxonomies recognise 100 genera comprising more
than 700 species (Kurtzman & Fell, 1998b), of which approxi-
mately 20 are relevant to winemaking (Fleet, 1993). Yeast genera,
with those non-Saccharomyces yeasts relevant to winemaking
indicated in bold type, are listed in Table 1.
Rules for taxonomy of yeasts fall under the authority of the
International Code of Botanical Nomenclature (Greuter et al.,
1994). Publication of new species must include a description of
essential characteristics, as well as a diagnosis that distinguishes
S. Afr. J. Enol. Vitic., Vol. 27, No. 1, 2006
16
the taxon from previously described species. Names of taxa must
be given in Latin or modified in such a way that they follow the
rules of Latin derivation, including appropriate designations.
The first level of yeast classification is based on the lack of a
sexual phase during the life cycle (Deuteromycotina) or aspects
of the sexual phase (Ascomycotina and Basidiomycotina).
Further taxonomic subdivisions (orders, families, genera and
species) are based on morphological, physiological, biochemical
and genetic properties (Kreger-van Rij, 1984; Kurtzman & Fell,
1998b) that are elucidated by conducting 55 to 70 tests. Many of
these tests can be used individually to characterise a selection of
yeasts.
Some yeasts are found as a sexual (teleomorphic) type and pro-
duce ascospores. A similar form of the same yeast is the asexual
(anamorphic) type that does not form ascospores. To complicate
matters, the ability to form ascospores can be lost during long-
term storage (Yarrow, 1998; M. Th. Smith, personal communica-
tion, 2000). Sporulation is also difficult to induce for some yeasts.
Whether a newly isolated yeast is subsequently identified as
teleomorphic or anamorphic can therefore largely depend on the
time lapsed between isolation and identification as well as adher-
ence to methodology. This can lead to confusion when authors
report on isolates as essentially the same yeast species, but refer
to them by different names. If any uncertainty exists in determin-
ing sporulation it is therefore preferable to use the anamorphic
name where applicable. Some of the more commonly encoun-
tered anamorphic yeasts and their teleomorphic counterparts in
must and wine are given in Table 2.
Teleomorphic
ascomycetous genera
(Ascomycotina)
Anamorphic
ascomycetous genera
(Deuteromycotina)
Teleomorphic
heterobasidio-mycetous genera
(Basidiomycotina)
Anamorphic
heterobasidio-mycetous genera
(Basidiomycotina)
Ambrosiozyma Aciculoconidium Agaricostilbum Bensingtonia
Arxiozyma Arxula Bulleromyces Bullera
Ascoidea Blastobotrys Chionosphaera Cryptococcus
Babjevia Botryozyma Cystofilobasidium Fellomyces
Cephaloascus Brettanomyces Erythrobasidium Hyalodendron
Citeromyces Candida Fibulobasidium Itersonilia
Clavispora Geotrichum Filobasidiella Kockovaella
Coccidiascus Kloeckera Filobasidium Kurtzmanomyces
Cyniclomyces Lalaria Holtermannia Malassezia
Debaryomyces Myxozyma Leucosporidium Moniliella
Dekkera Oosporidium Mrakia Phaffia
Dipodascopsis Saitoella Rhodosporidium Pseudozyma
Dipodascus Schizoblastosporion Sirobasidium Reniforma
Endomyces Sympodiomyces Sporidiobolus Rhodotorula
Eremothecium Trigonopsis Sterigmatosporidium Sporobolomyces
Galactomyces Tilletiaria Sterigmatomyces
Hanseniaspora Tremella Sympodiomycopsis
Issatchenkia Trimorphomyces Tilletiopsis
Kluyveromyces Xanthophyllomyces Trichosporon
Lipomyces Trichosporonoides
Lodderomyces Tsuchiyaea
Metschnikowia
Nadsonia
Pachysolen
Pichia
Protomyces
Saccharomyces
Saccharomycodes
Saccharomycopsis
Saturnispora
Schizosaccharomyces
Sporopachydermia
Stephanoascus
Torulaspora
Wickerhamia
Wickerhamiella
Williopsis
Yarrowia
Zygoascus
Zygosaccharomyces
Zygozyma
1
Non-Saccharomyces genera that can be encountered in vineyards, on winery surfaces, in grape musts and/or in wine are indicated in bold type.
TABLE 1
A list of yeast genera
1
according to Kurtzman & Fell (1998b).
17
ECOLOGY OF YEASTS
Yeasts are found throughout nature. However, they do not occur
randomly, but are found in specific habitats where different
species form communities (Lachance & Starmer, 1998). The dif-
ferent species found in a habitat can either be autochothonous
(those that are essential components of the community) or allo-
chothonous (those that are transient, or there by chance). The com-
ponent species within yeast communities are further defined by
niches, i.e. the physical, chemical and biotic attributes required by
the yeast to survive and grow (Lachance & Starmer, 1998).
Yeasts found in many different habitats are considered general-
ists (broad niche), while those found in unique habitats are con-
sidered specialists (narrow niche) (Lachance & Starmer, 1998).
Within the winemaking environment (habitat), the vineyard
(grape surfaces) and cellar (equipment surfaces and must) can be
considered specialised niches where the wine related yeasts can
form communities (Polsinelli et al., 1996). These niches differ
broadly. The surface of the grape berry before ripeness presents
limitations regarding nutrients. These are alleviated as berries
ripen and/or are damaged. External factors such as fungicides and
pesticides used in the vineyard will have a negative impact on
populations. However, it has been reported that some pesticides
can stimulate certain yeasts, e.g. K. apiculata, when tested in lab-
oratory fermentations (Cabras et al., 1999).
Grape must is a rich nutritive environment, but low pH and high
osmotic pressure of the must and the use of SO2 detracts from this
otherwise ideal yeast niche. Surfaces of cellar equipment can also
harbour numerous microorganisms due to constant contact with
grape must. Cellar hygiene consequently plays a big role in this
niche.
NON-SACCHAROMYCES YEASTS ASSOCIATED WITH
GRAPES, FERMENTING MUST AND WINE
The yeast species found in different niches associated with grape
growth (vineyards) and wine production (wineries, grape must,
fermentation and wine) can be arbitrarily divided into two groups,
i.e. the Saccharomyces group and the non-Saccharomyces group.
The Saccharomyces group with its primary representative,
Saccharomyces cerevisiae, is present on grape skins in low num-
bers (Van Zyl & Du Plessis, 1961; Rankine, 1972; Trk et al.,
1996), and on winery equipment and in fermenting must in
greater numbers (Peynaud & Domercq, 1959; Vaughan-Martini &
Martini, 1995). S. cerevisiae is the most important yeast for wine
production and is responsible for the metabolism of grape sugar
to alcohol and CO2 (Reed & Peppler, 1973; Fleet, 1993; Pretorius
et al., 1999; Pretorius, 2003; Swiegers & Pretorius, 2005;
Swiegers et al., 2005). It has an equally important role to play in
the formation of secondary metabolites of importance to wine
(Fleet, 1993; Pretorius, 2003), as well as in the conversion of
grape aroma precursors to varietal aromas in wine (Darriet et al.,
1995; Dubourdieu, 1996; Ribreau-Gayon et al., 2000; Howell et
al., 2004). For these reasons S. cerevisiae is often simply referred
to as the wine yeast. The knowledge pertaining to S. cerevisiae
during wine fermentation can often be applied to non-
Saccharomyces yeasts under the same conditions and in the same
environment. In addition, as most fields of research are focussed
primarily on S. cerevisiae, non-Saccharomyces research can be-
TABLE 2
Anamorphs, teleomorphs and synonyms of some of the non-Saccharomyces yeasts in the Ascomycetous genera encountered in wine
fermentations (Kurtzman & Fell, 1998b).
Anamorphic form Teleomorphic form Synonyms
1
Brettanomyces bruxellensis Dekkera bruxellensis
Candida colliculosa Torulaspora delbrueckii Saccharomyces rosei
Candida famata Debaryomyces hansenii
Candida globosa Citeromyces matritensis
Candida guilliermondii Pichia guilliermondii
Candida hellenica Zygoascus hellenicus
Candida lambica Pichia fermentans
Candida pelliculosa Pichia anomala Hansenula anomala
Candida pulcherrima Metschnikowia pulcherrima Torulopsis pulcherrima
Candida reukaufii Metschnikowia reukaufii
Candida sorbosa Issatchenkia occidentalis
Candida stellata -
2
Torulopsis stellata
Candida valida Pichia membranifaciens
Kloeckera africana Hanseniaspora vineae
Kloeckera apiculata Hanseniaspora uvarum
Kloeckera apis Hanseniaspora guilliermondii
Kloeckera corticis Hanseniaspora osmophila
Kloeckera javanica Hanseniaspora occidentalis
-
3
Issatchenkia terricola Pichia terricola
-
3
Kluyveromyces thermotolerans
-
3
Saccharomyces kluyveri
-
3
Saccharomycodes ludwigii
-
3
Zygosaccharomyces bailii Saccharomyces bailii
-
3
Pichia farinosa
1
Names sometimes found in older literature.
2
No teleomorphic form.
3
No anamorphic form.
18
nefit from the techniques developed by the S. cerevisiae
researchers.
The non-Saccharomyces yeasts contain numerous species,
dominated numerically by the apiculate yeasts, e.g. Kloeckera
spp. and Candida spp. that are found predominantly on grapes
and in freshly processed must. Lesser numbers are found on win-
ery equipment.
The microflora of grapes is affected by a number of factors.
These include vineyard altitude and aspect, climatic conditions
(temperature, rainfall, humidity, maritime influences), grape vari-
ety (cultivar, thickness of grape skin), viticultural practices (fer-
tilisation, irrigation, canopy management, use of fungicides, use
of elemental sulphur), developmental stage of grapes, health of
grapes (physical damage to berries, insect pests) and winery
waste-disposal practices (Bisson & Kunkee, 1991; Regueiro et
al., 1993; Boulton et al., 1996; Epifanio et al., 1999; Pretorius,
2000). The manner in which grapes are sampled (e.g. the berries
or bunches) and processed (washing vs. crushing) can also deter-
mine what yeasts are isolated (Martini et al., 1980; 1996), as the
number of yeast cells is greater close to the peduncle than it is at
the centre and lower part of the bunch (Rosini et al., 1982).
At harvest, grape temperature, method of harvest (manual vs.
mechanical), method of transport to the cellar (picking crates/bas-
kets, tipsters), time of transport to the cellar, time lapse before
crushing, and sulphite and enzyme addition can all affect yeast
populations (Pretorius et al., 1999; Pretorius, 2000). Yeasts found
on the surface of grapes are introduced into the must at crushing
(Bisson & Kunkee, 1991; Lonvaud-Funel, 1996). Other strains
found on the surface of cellar equipment can also be transferred
to the must (Boulton et al., 1996). Populations are further affect-
ed by the method of crushing, i.e. pressing whole bunches vs.
berries, sulphite addition, enzyme addition, cellar hygiene, type
of equipment used, clarification method and temperature control
(Regueiro et al., 1993; Epifanio et al., 1999; Pretorius et al.,
1999; Pretorius, 2000).
The specific environmental conditions in the must, i.e. high
osmotic pressure, presence of SO2, and temperature, all play a
role in determining what species can survive and grow (Bisson &
Kunkee, 1991; Longo et al., 1991). The fermentation rapidly
becomes anaerobic and the alcohol levels increase, which further
affects yeast populations.
Despite all the variables in grape harvest and wine production,
the yeast species generally found on grapes and in wines are sim-
ilar throughout the world (Amerine et al., 1967). However, the
proportion (or population profile) of yeasts in the different
regions shows distinct differences (Amerine et al., 1967; Longo
et al., 1991).
In areas with high rainfall during harvest the numbers of non-
Saccharomyces yeasts increase (Querol et al., 1990). Pesticides
and other chemical sprays used in the vineyard can also affect
yeast populations (Monteil et al., 1987; Cabras et al., 1999;
Guerra et al., 1999).
The range of non-Saccharomyces species isolated often
depends on the place from which, and the stage in the winemak-
ing process at which, the samples are taken (see Tables 3 & 4).
The methods of isolation and enumeration can also impact on the
type of yeasts that are isolated, as evident from Table 3. Such
methods include shaking grape berries in a broth or crushing
whole berries and plating on nutrient agar media. The technique
of crushing berries before plating is closer to practical winemak-
ing protocols than is shaking in a broth, but the objective of the
investigation will determine which method is chosen.
The type of growth medium used can also play an important
role by limiting the growth of specific yeasts. The use of Lysine
Medium, for example, does not allow the growth of S. cerevisiae
due to the inability of this yeast to utilise lysine as the sole car-
bon source (Fowell, 1965; Heard & Fleet, 1986). However, some
non-Saccharomyces yeasts might also not be able to utilise lysine
and, therefore, will not be detected. On a general nutrient agar
medium, fast-growing yeasts can also overgrow slow growers.
For yeasts with similar growth rates, only yeasts present in the
same numerical order will be detected, and more specific tech-
niques and media are needed to isolate slower growing yeasts or
yeasts found in low numbers.
The aforementioned limitations can be overcome to a degree by
using culture-independent techniques. These include the use of
epi-fluorescence microscopy (Du Toit et al., 2005), PCR (poly-
merase chain reaction) based DGGE (denaturing gradient gel
electrophoreses) (Cocolin et al., 2000; Prakitchaiwattana et al.,
2004) and FT-IR (Fourier-transform infrared) spectroscopy
(Wenning et al., 2002).
Non-Saccharomyces yeasts in vineyards and on grapes
Low numbers of yeasts (10-10
3
cfu/g) are found on unripe grapes,
but as the grapes ripen the numbers increase to 10
4
-10
6
cfu/g
(Fleet, 2003). This is due to sugars that leach or diffuse out from
inner tissue to the grape skin surfaces, providing nutrition for the
yeasts. Damaged berries increase the leaching effect. Therefore,
the maturity of the grapes and/or the degree of damage to grape
berries will largely determine the population numbers.
Generally, between nine and 15 culturable yeast species are
found on grapes (Du Plessis, 1959; Van Zyl & Du Plessis, 1961;
Parish & Caroll, 1985; Yanagida et al., 1992; Regueiro et al.,
1993; Zahavi et al., 2002; Jolly et al., 2003a; Rementeria et
al., 2003). These include Hanseniaspora/Kloeckera spp.,
Metschnikowia/Candida spp., Rhodotorula spp. and Cryptococ-
cus spp. Unfortunately, comparisons between different studies are
difficult, as different approaches have been used for grape sam-
pling and yeast isolation (see Table 3). In addition, the state of
ripeness and berry damage is never given. Further factors that can
influence a yeast population include specific meso- and micro-
climates in vineyards. Notwithstanding, there is general agree-
ment that the most frequently occurring species in vineyards are
usually the apiculate yeasts, K. apiculata/Hanseniaspora uvarum
(50-75% of isolates) (Van Zyl & Du Plessis, 1961; Yanagida et
al., 1992; and a recent review by Pretorius, 2000). It has been
reported that in warm to hot regions the teleomorphic form
(H. uvarum) tends to replace the anamorphic form (K. apiculata),
while the anamorphic form is present in greater numbers in cool-
er regions (Bisson & Kunkee, 1991; Jackson, 1994; Boulton et
al., 1996). In moderate climates both types occur in equal num-
bers. However, this distribution between the teleomorphic and
anamorphic forms might be region dependent. Altitude also
appears to play a role as it has been reported that Kloeckera spp.
are found more frequently at high altitudes and Hansenia-
19
spora spp. more frequently at low altitudes. This might be linked
to temperature. Identification of Kloeckera vs. Hanseniaspora
yeasts also depends on how long the yeasts have been conserved
before identification (Yarrow, 1998; M. Th. Smith, personal com-
munication, 2000).
According to Van Zyl & Du Plessis (1961), the following yeasts
occurred in highest frequency in South African vineyards:
K. apiculata, Rhodotorula glutinis, Candida krusei, Candida pul-
cherrima, Candida laurentii, Cryptococcus albidus, and Candida
stellata (T. bacillaris). In a more recent, but limited, investigation
(Jolly et al., 2003a), K. apiculata, C. pulcherrima and a
Rhodotorula sp. were still found in dominant numbers but
Kluyveromyces thermotolerans and Zygosaccharomyces bailii
were also isolated. Studies by Le Roux et al. (1973) showed that
Botrytis cinerea infection of grapes influenced the
non-Saccharomyces populations C. krusei and K. apiculata
increased and R. glutinis decreased. Other non-Saccharomyces
yeasts found on grapes and in vineyards are shown in Table 3.
Non-Saccharomyces yeasts associated with fermenting must
During crushing, the non-Saccharomyces yeasts on the grapes, on
cellar equipment and in the cellar environment (air- and insect-
borne) are carried to the must (Peynaud & Domercq, 1959;
Bisson & Kunkee, 1991; Boulton et al., 1996; Lonvaud-Funel,
1996; Trk et al., 1996; Constant et al., 1997; Mortimer &
Polsinelli, 1999; Fleet, 2003). Non-Saccharomyces species that
have been isolated from cellar surfaces include Pichia anomala,
Pichia membranifaciens, Candida spp., Cryptococcus spp. and,
more rarely, Rhodotorula spp., Debaryomyces hansenii, K. apic-
ulata and Metschnikowia pulcherrima (Loureiro & Malfeito-
Ferreira, 2003). However, cellar surfaces play a smaller role than
grapes as a source of non-Saccharomyces yeasts, as S. cerevisiae
is the predominant yeast inhabiting such surfaces (Peynaud &
Domercq, 1959; Rosini, 1984; Lonvaud-Funel, 1996; Pretorius,
2000). Furthermore, hygienic procedures used in most modern
cellars should minimise contamination of must by resident cellar
flora (Jackson, 1994; Pretorius, 2000). It might therefore be
expected that the dominant yeasts in must after crushing will be
the same as are found on grapes (Rementeria et al., 2003).
The specific environmental conditions in grape must are limit-
ing and hostile to yeasts due to low pH, high sugar (high osmot-
ic pressure), an equimolar mixture of glucose and fructose, pres-
ence of SO2 and a non-optimal growth temperature during cold
settling (Bisson & Kunkee, 1991; Longo et al., 1991; Pretorius,
2000). Furthermore, the environment rapidly becomes anaerobic,
with increasing levels of ethanol that is toxic to yeasts. Nitrogen
levels are usually sufficient at the start of fermentation (Bisson &
Kunkee, 1991), but can be limiting towards the end of fermenta-
tion unless supplemented. The clarification of white must (cen-
trifugation, enzyme treatments, cold settling) can also reduce the
initial population of yeasts (Fleet, 1990; Lonvaud-Funel, 1996;
Pretorius, 2000).
Non-Saccharomyces yeasts found in grape must and during fer-
mentation (see Table 4) can be divided into three groups:
(i) yeasts that are largely aerobic, e.g. Pichia spp., Debaryo-
myces sp., Rhodotorula spp., Candida spp. (e.g. C. pul-
cherrima and C. stellata), and Cryptococcus albidus;
(ii) apiculate yeasts with low fermentative activity, e.g.
K. apiculata (H. uvarum), Kloeckera apis, Kloeckera
javanica; and
(iii) yeasts with fermentative metabolism, e.g. Kluyveromyces
marxianus, Torulaspora spp. (e.g. T. globosa and T. del-
brueckii) and Zygosaccharomyces spp. (Fleet et al., 1984;
Querol et al., 1990; Bisson & Kunkee, 1991; Longo et al.,
1991; Lonvaud-Funel, 1996; Lorenzini, 1999; Torija et
al., 2001; Combina et al., 2005).
During fermentation, and especially in spontaneous fermenta-
tions, there is a sequential succession of yeasts. Initially, species
of Kloeckera (Hanseniaspora), Rhodotorula, Pichia, Candida
(C. stellata, C. pulcherrima [M. pulcherrima], Candida sake) and
Cryptococcus are found at low levels in the fresh must (Parish &
Caroll, 1985; Bisson & Kunkee, 1991; Frezier & Dubourdieu,
1992; Jackson, 1994; Granchi et al., 1998; Fleet, 2003; Combina
et al., 2005). Of these, K. apiculata is usually present in the high-
est numbers, followed by various Candida spp.
In a study of South African musts, however, very few K. apic-
ulata yeasts were found (Van Zyl & Du Plessis, 1961). This was
attributed to the addition of large quantities of SO2 to the must to
aid settling. In another study on muscadine (Vitis rotundifolia)
grapes from North Carolina H. uvarum (K. apiculata) was absent,
but Hanseniaspora osmophilia and P. membranifaciens predomi-
nated during the initial stages of the fermentation (Parish &
Caroll, 1985). This might be an association for that particular
geographic area or grape type. However, viticultural practices
could have affected the normal non-Saccharomyces population.
At the start of fermentation an initial proliferation of apiculate
yeasts (Kloeckera and Hanseniaspora) normally occurs. This is
usually more apparent in red must than in white, possibly due to
the higher pH of the former.
In the past it was generally believed that all non-Saccharomyces
yeasts died soon after the commencement of an alcoholic fer-
mentation due to the increasing ethanol concentration and added
SO2. However, more recent work has shown that some
non-Saccharomyces yeasts can survive to a later stage of fermen-
tation (up to 12 days) than initially believed (Fleet et al., 1984;
Heard & Fleet, 1985; Fleet, 1990; Longo et al., 1991; Todd, 1995;
Gafner et al., 1996; Granchi et al., 1998; Zohre & Erten, 2002;
Fleet, 2003; Combina et al., 2005). Other non-Saccharomyces
yeasts might be present throughout the fermentation, reaching
cell densities of 10
6
to 10
8
cells/mL (Fleet et al., 1984; Combina
et al., 2005). This sustained growth of non-Saccharomyces spp. is
more evident in spontaneous fermentations, which lack the initial
high density inoculum of S. cerevisiae. Abnormal vintages due,
for example, to excessive rainfall during grape ripening, also con-
tribute to greater numbers of non-Saccharomyces yeasts in the
initial stages and later in the fermentation (Querol et al., 1990).
Non-Saccharomyces yeasts have also been observed to grow to
levels of ca. 10
4
cells/mL in red wines during malo-lactic fer-
mentations (Fleet et al., 1984).
Despite the sustained presence of certain non-Saccharomyces
yeasts, the majority disappear during the early stages of a vigor-
ous fermentation (Fleet et al., 1984; Jackson, 1994; Henick-Kling
et al., 1998). This might be due to their slow growth and inhibi-
tion by the combined effects of SO2, low pH, high ethanol content
and oxygen deficiency (Jackson, 1994; Combina et al., 2005).
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Candida albicans North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
(Vitis rotundifolia)
Candida edax / Stephanoascus smithiae A North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
Candida famata / Debaryomyces hansenii A Israel Muscat d Alexandrie Twenty to fifty berries shaken in sterile distilled water and plated onto basal yeast agar Zahavi et al., 2002
A Israel Cabernet Sauvignon Twenty to fifty berries shaken in sterile distilled water and plated onto basal yeast agar Zahavi et al., 2002
Candida glabrata [Torulopsis glabrata] S Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Candida globosa / Citeromyces matritensis T Israel Cabernet Sauvignon, Twenty to fifty berries shaken in sterile distilled water and plated onto basal yeast agar Zahavi et al., 2002
Colombard
Candida guilliermondii / A Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Pichia guilliermondii or Pichia ohmeri A Israel Cabernet Sauvignon Twenty to fifty berries shaken in sterile distilled water and plated onto basal yeast agar Zahavi et al., 2002
Candida inconspicua S Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
[Torulopsis inconspicua]
Candida krusei / Issatchenkia orientalis A Western Cape Grapes Berries incubated in sterile grape juice until growth observed and then plated on Van Zyl & Du Plessis,
malt extract and grape juice agar medium 1961
Candida lambica / Pichia fermentans T Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Candida melinii / Pichia canadensis A Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Candida pulcherrima / Metschnikowia A Western Cape Grapes Berries incubated in sterile grape juice until growth observed and then plated on Van Zyl & Du Plessis,
pulcherrima [Torulopsis pulcherrima] malt extract and grape juice agar medium 1961
A Israel Cabernet Sauvignon Twenty to fifty berries shaken in sterile distilled water and plated onto basal yeast agar Zahavi et al., 2002
Candida reukaufi / T Israel Muscat d Alexandrie Twenty to fifty berries shaken in sterile distilled water and plated onto basal yeast agar Zahavi et al., 2002
Metschnikowia reukaufi
Candida sake North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
Candida stellata [Torulopsis bacillaris] S Western Cape Grapes Berries incubated in sterile grape juice until growth observed and then plated on Van Zyl & Du Plessis,
S Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Candida valida / Pichia membranifaciens T North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
Candida vini [Candida mycoderma] S Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Candida zeylanoides Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Van Zyl & Du Plessis,
1961
Cryptococcus albidus Western Cape Grapes Berries incubated in sterile grape juice until growth observed and then plated on Van Zyl & Du Plessis,
[Cryptococcus diffluens] malt extract and grape juice agar medium 1961
Japan Zenkoji and Koshu Berries crushed; dilutions plated Yanagida et al., 1992
Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
S Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Yeast species
Anamorph / Teleomorph
[Synonym]
1
Region or
country
Isolation material Brief description of method of isolation Reference
F
o
r
m

i
s
o
-
l
a
t
e
d

A
/
T
/
S
2
TABLE 3
Non-Saccharomyces yeasts isolated from grapes.
S
.

A
f
r
.

J
.

E
n
o
l
.

V
i
t
i
c
.
,
V
o
l
.

2
7
,
N
o
.

1
,
2
0
0
6
2
1
T
h
e

R
o
l
e

a
n
d

U
s
e

o
f

N
o
n
-
S
a
c
c
h
a
r
o
m
y
c
e
s

Y
e
a
s
t
s

i
n

W
i
n
e

P
r
o
d
u
c
t
i
o
n
Cryptococcus humicolus Spain Traditional Ten berries washed in saline and plated on Sabouraud dextrose agar Rementeria et al., 2003
[Candida humicola] grape varieties
S North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
Cryptococcus laurentii Western Cape Grapes Berries incubated in sterile grape juice until growth observed and then plated on Van Zyl & Du Plessis,
Japan Chardonnay, Zenkoji Berries crushed; dilutions plated Yanagida et al., 1992
and Koshu
Cryptococcus neoformans / A Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Filobasidiella neoformans
Kloeckera apiculata / A Western Cape Grapes Berries incubated in sterile grape juice until growth observed and then plated on Van Zyl & Du Plessis,
Hanseniaspora uvarum malt extract and grape juice agar medium 1961
A Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
A Japan Niagra, Zenkoji Berries crushed; dilutions plated Yanagida et al., 1992
and Koshu
T California Vineyard Fermentations at 13C and 18C; dilutions plated on WL medium Pallmann et al., 2001
Kloeckera corticis / T North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
Hanseniaspora osmophila (Vitis rotundifolia)
[K. magna]
Kloeckera javanica / T Japan Niagra Berries crushed; dilutions plated Yanagida et al., 1992
Hanseniaspora occidentalis
Lodderomyces elongisporus North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
Rhodotorula aurantiaca Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
Rhodotorula glutinus North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
Western Cape Grapes Berries incubated in sterile grape juice until growth observed and then plated on Van Zyl & Du Plessis,
Japan Chardonnay and Berries crushed; dilutions plated Yanagida et al., 1992
Zenkoji
Spain Traditional grape Ten berries washed in saline and plated on Sabouraud dextrose agar Rementeria et al., 2003
varieties
Rhodotorula minuta North Carolina Muscadine Ten berries shaken in peptone buffer and plated on potato dextrose agar Parish & Carroll, 1985
Japan Zenkoji Berries crushed; dilutions plated Yanagida et al., 1992
Rhodotorula mucilaginosa Western Cape Grapes Berries shaken in sterile distilled water with Tween 80 and plated on grape must agar Le Roux et al., 1973
1
Where applicable, the anamorph and teleomorph designations are given, and also the synonym or alternate name used in older literature.
Yeast nomenclature according to Kurtzman & Fell (1998).
2
A = anamorph; T = teleomorph; S = synonym.
Yeast species
[Synonym]
1
Region or
country
Isolation material Brief description of method of isolation Reference
F
o
r
m

i
s
o
-
l
a
t
e
d

A
/
T
/
S
2
TABLE 3 (continued)
Non-Saccharomyces yeasts isolated from grapes.
22
This is consistent with their oxidative or weak fermentative
metabolism. Nutrient limitation and size of S. cerevisiae inocu-
lum would also have a suppressive effect. Granchi et al. (1998)
reported that numbers of K. apiculata declined once S. cerevisiae
became dominant rather than when the fermentation temperature
and ethanol concentration reached values known to inhibit apicu-
late yeast growth. It has also been reported that T. delbrueckii and
K. thermotolerans are less tolerant to low oxygen levels and it is
this, rather than ethanol toxicity, that affects their growth and
leads to their death during fermentation (Hansen et al., 2001). It
was also shown that a cell-cell contact mechanism in the presence
of high concentrations of viable S. cerevisiae yeasts played a role
in the inhibition of these two non-Saccharomyces species (Nissen
et al., 2003).
The non-Saccharomyces spp. that survive and are present until
the end of fermentation may also have a higher tolerance to
ethanol. It has been documented that C. stellata (Torulopsis stel-
lata) can tolerate up to 12% ethanol (Combina et al., 2005),
which would account for its sustained presence during fermenta-
tion. Other species reported throughout fermentation are Z. bailii
(Saccharomyces acidifaciens) (Peynaud & Domercq, 1959) and
Pichia sp. (Bisson & Kunkee, 1991).
The extent to which different factors affect the non-
Saccharomyces yeasts are dependent on the characteristics of the
individual species. Growth parameters for one species will not
necessarily be the same for others. Variations can also occur for
strains within a species.
Non-Saccharomyces yeasts associated with wine
Non-Saccharomyces yeasts in wine are usually associated with
wines in barrels and post-fermentation spoilage (Van der Walt &
Van Kerken, 1958; Amerine & Cruess, 1960; Van Zyl, 1962;
Heresztyn, 1986; Grbin, 1999; and a recent review by Loureiro &
Malfeito-Ferreira, 2003). However, only a small number are able
to tolerate the adverse conditions in wine, and multiply (Van
Kerken, 1963). These include Brettanomyces spp. (Dekkera spp.),
Z. bailii, P. membranifaciens, C. krusei and C. valida (Van
Kerken, 1963; Fleet et al., 1984; Parish & Caroll, 1985; Bisson &
Kunkee, 1991; Grbin, 1999). Some of these species, e.g.
Brettanomyces spp. and Zygosaccharomyces spp. are as ethanol
tolerant as S. cerevisiae and may be found in bottled wine. Their
presence is influenced by the degree of filtration that precedes
bottling and cellar hygiene during bottling.
THE ROLE OF NON-SACCHAROMYCES YEASTS IN WINE
PRODUCTION
The role of non-Saccharomyces yeasts in wine production has
been debated extensively (Castor, 1954; Van Zyl et al., 1963;
Fleet et al., 1984; Heard & Fleet, 1985; Fleet, 1990; Herraiz et
al., 1990; Longo et al., 1991; Romano et al., 1992; Todd, 1995;
Gafner et al., 1996; Gil et al., 1996; Lema et al., 1996; Granchi
et al., 1998; Henick-Kling et al., 1998; Lambrechts & Pretorius,
2000; Fleet, 2003; Rementeria et al., 2003; Combina et al., 2005).
As already discussed, grape musts contain a mixture of yeast
species. Wine fermentation is therefore not a single-species fer-
mentation (Fleet, 1990), although the dominance of S. cerevisiae
(inoculated or indigenous) in the fermentation is expected and
desired. However, the indigenous non-Saccharomyces yeasts,
already present in the must, and often in greater numbers than
S. cerevisiae, are adapted to the specific environment and are in
an active growth state, giving them a competitive edge.
Despite a long-held belief among winemakers in Old World
wine regions that spontaneous fermentations (comprising mixed
cultures of non-Saccharomyces and Saccharomyces yeasts) pro-
duce superior wines compared with pure culture fermentations,
earlier authors usually refer to the non-Saccharomyces yeasts as
spoilage organisms or wild yeasts (Amerine & Cruess, 1960;
Van Zyl & Du Plessis, 1961; Van Kerken, 1963; Rankine, 1972;
Le Roux et al., 1973). This was substantiated by their frequent
isolation from stuck fermentations and from spoiled bottles of
wine.
Furthermore, although it was known that some non-
Saccharomyces yeasts could form metabolites, e.g. esters, leading
to aromas not always detrimental to wine quality (Castor, 1954;
Amerine & Cruess, 1960; Van Zyl et al., 1963), this was out-
weighed by the high levels of volatile acids and other undesirable
compounds produced (Castor, 1954; Amerine & Cruess, 1960;
Van Zyl et al., 1963; Amerine et al., 1967; 1972). Some yeasts,
e.g. Candida, Pichia and Hansenula spp. are capable of forming
films on the surface of wine exposed to oxygen. Off-odours,
including acetic acid, ethyl acetate and acetaldehyde are also
associated with their growth (Grbin, 1999). Brettanomyces spp.
(Dekkera spp.) can contribute to animal/farmyard/mousy taints
in wines (Parish & Caroll, 1985; Grbin, 1999, Grbin & Henschke,
2000; Arvik & Henick-Kling, 2002; Du Toit et al., 2005). It has
also been reported that Brettanomyces bruxellensis can form bio-
genic amines (Caruso et al., 2002) that can lead to undesirable
physiological effects in sensitive humans. Other non-
Saccharomyces yeasts such as Saccharomycodes ludwigii, more
commonly a contaminant of sulphated musts due to its high resis-
tance to SO2, produce large amounts of ethyl acetate and
acetaldehyde that negatively affect wine aroma and quality (Ciani
& Maccarelli, 1998).
Authors of earlier publications also considered non-
Saccharomyces yeasts to be sensitive to SO2 in must and added
SO2 primarily to control their growth and that of spoilage bacte-
ria (Amerine & Cruess, 1960; Van Zyl & Du Plessis, 1961;
Amerine et al., 1972). Non-Saccharomyces yeasts were also
known to be poor fermenters of grape must and intolerant to
ethanol (Castor, 1954), especially in the presence of SO2
(Amerine et al., 1972). It was therefore accepted that those non-
Saccharomyces yeasts not initially inhibited by the SO2 died dur-
ing fermentation due to the combined toxicity of the SO2 and
alcohol. Consequently, the non-Saccharomyces yeasts were seen
to be of little significance in normal wine production and it was
recommended that only proven strains of the wine yeast S. cere-
visiae be used in commercial fermentations (Amerine & Cruess,
1960; Amerine et al., 1972).
As already mentioned, non-Saccharomyces yeasts can survive
and reach high cell densities, similar to S. cerevisiae (10
6
to 10
8
cells/mL), during fermentation. More recently reported higher
numbers of non-Saccharomyces yeasts might be the result of
improved cellar technology and hygiene in modern cellars that
has led to a reduction in SO2 usage, presumably resulting in the
survival of a greater number and diversity of non-Saccharomyces
yeasts. Coupled to this is the use of modern laboratory techniques
that makes the detection of non-Saccharomyces yeasts easier.
S
.

A
f
r
.

J
.

E
n
o
l
.

V
i
t
i
c
.
,
V
o
l
.

2
7
,
N
o
.

1
,
2
0
0
6
2
3
T
h
e

R
o
l
e

a
n
d

U
s
e

o
f

N
o
n
-
S
a
c
c
h
a
r
o
m
y
c
e
s

Y
e
a
s
t
s

i
n

W
i
n
e

P
r
o
d
u
c
t
i
o
n
Brettanomyces bruxellensis / Dekkera T Tenerife White Listan Random harvesting; further details not given De Cos et al., 1999
bruxellensis [Brettanomyces vini]
A Bordeaux Red variety Not given Peynaud & Domercq, 1959
T Spain Ribeiro Dilution and plating on YPD Cansado et al., 1989
Candida sp. Spain Ribeiro Dilution and plating on YPD Cansado et al., 1989
Candida colliculosa /
Torulaspora delbrueckii [Torulaspora rosei]
[Saccharomyces fermentati] A Australia Hermitage (red) Spread inoculation on malt extract and Lysine agar Heard & Fleet, 1985
A Catalonia Macabeo (white) and Grenache (red) Dilution and plating on YPD Torija et al., 2001
T Bordeaux White variety Not given Peynaud & Domercq, 1959
T Italy Variety not given Not given Castelli, 1955
T Tenerife White Listan Random harvesting; further details not given De Cos et al., 1999
S Bordeaux Merlot Dilutions plated on malt extract agar & grape juice agar Fleet et al., 1984
T Bordeaux White variety Not given Peynaud & Domercq, 1959
Candida famata / T Tenerife White Listan Random harvesting; further details not given De Cos et al., 1999
Debaryomyces hansenii
A Israel Muscat d Alexandrie Twenty to fifty berries shaken in sterile distilled
water and plated on basal yeast agar Zahavi et al., 2002
T Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
T Bordeaux Red variety Not given Peynaud & Domercq, 1959
Candida glabrata Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Candida glucosophila Spain White and red (traditional varieties) Samples plated on Sabouraud dextrose agar Rementeria et al., 2003
Candida guilliermondii / A Spain Abarino, Godello(white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Pichia guilliermondii or Pichia ohmeri
Candida hellenica / Zygoascus
hellenicus [Candida steatolytica] S Majorca Prensal blanc (white) Dilutions plated onto YM and lysine agar Mora & Mulet, 1991
Candida kefyr / Kluyveromyces marxianus T Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Candida krusei / Issatchenkia orientalis A Bordeaux Semillon Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
[Saccharomyces krusei] S Bordeaux Merlot Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
T Argentina Malbec Plated on malt extract agar Combina et al., 2005
Candida lambica / Pichia fermentans T Bordeaux Red and white varieties Not given Peynaud & Domercq, 1959
Candida lusitaneae / Clavispora lusitaneae A Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Candida pelliculosa / Pichia anomala
[Hansenula anomala] S Australia Hermitage (red) Spread inoculation on malt extract and lysine agar Heard & Fleet, 1985
S Bordeaux Red variety Not given Peynaud & Domercq, 1959
S Majorca Chenin blanc Dilutions plated on YM and lysine agar Mora & Mulet, 1991
Yeast species
[Synonym]
1
Country
or region
Isolation material
(must variety)
Brief description of method of isolation Reference
F
o
r
m

i
s
o
-
l
a
t
e
d

A
/
T
/
S
2
TABLE 4
Non-Saccharomyces yeasts isolated from grape must.
S
.

A
f
r
.

J
.

E
n
o
l
.

V
i
t
i
c
.
,
V
o
l
.

2
7
,
N
o
.

1
,
2
0
0
6
2
4
T
h
e

R
o
l
e

a
n
d

U
s
e

o
f

N
o
n
-
S
a
c
c
h
a
r
o
m
y
c
e
s

Y
e
a
s
t
s

i
n

W
i
n
e

P
r
o
d
u
c
t
i
o
n
Candida pulcherrima / Metschnikowia
pulcherrima [Torulopsis pulcherrima] A Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
T Catalonia Macabeo (white) and Grenache (red) Dilution and plating on YPD Torija et al., 2001
A Australia Riesling (white) and Malbec (red) Spread inoculation on malt extract and lysine agar Heard & Fleet, 1985
A Bordeaux Red grape variety Not given Peynaud & Domercq, 1959
T Bordeaux Semillon Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
A Majorca Prensal blanc (white) Dilutions plated on YM and lysine agar Mora & Mulet. 1991
T Italy Nebbiola (red) Dilutions plated on Phytone yeast extract agar Schtz & Gafner, 1993
T Germany Pinot noir (red) Dilutions plated on Phytone yeast extract agar Schtz & Gafner, 1993
S Switzerland Pinot noir (red) Dilutions plated on Phytone yeast extract agar Schtz & Gafner, 1993
T Italy Variety not given Details not given Castelli, 1955
A Argentina Malbec Plated on malt extract agar Combina et al., 2005
Candida rugosa Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Candida stellata [Torulopsis stellata; Australia Riesling, Semillon (white),
Torulopsis bacillaris] Malbec and Hermitage (red) Spread inoculation on malt extract and lysine agar Heard & Fleet, 1985
Catalonia Garnatxa Dilutions plated on malt extract agar Constant et al., 1997
Alicante Monastrell (red) Dilutions plated on malt extract agar Querol et al., 1990
S Bordeaux Merlot Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
S Bordeaux Semillon Dilutions plated on malt extract agar Fleet et al., 1984
Catalonia Macabeo (white) & Grenache (red) Dilution and plating on YPD Torija et al., 2001
S Bordeaux Red & white variety Not given Peynaud & Domercq, 1959
Majorca Chenin blanc & Prensal blanc Dilutions plated on YM and lysine agar Mora & Mulet, 1991
Argentina Malbec Plated on malt extract agar Combina et al., 2005
Candida valida / Pichia membranifaciens T Tenerife White Listan Random harvesting; further details not given. De Cos et al., 1999
T Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
T North Carolina Muscadine (Vitis rotundifolia) Direct plating on potato dextrose agar Parish & Carroll, 1985
T Alicante Monastrell (red) Dilutions plated on malt extract agar Querol et al., 1990
T Majorca Prensal blanc Dilutions plated on YM and lysine agar Mora & Mulet, 1991
T Bordeaux Red and white varieties Not given Peynaud & Domercq, 1959
T Argentina Malbec Plated on malt extract agar Combina et al., 2005
Candida vini Argentina Malbec Plated on malt extract agar Combina et al., 2005
Debaryomyces etchellsii [Pichia etchelsii] S Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Issatchenkia terricola Majorca Chenin blanc Dilutions plated on YM and lysine agar Mora & Mulet, 1991
Kloeckera sp. Spain Ribeiro Dilution and plating on YPD Cansado et al., 1989
Yeast species
[Synonym]
1
Country
or region
Isolation material
(must variety)
F
o
r
m

i
s
o
-
l
a
t
e
d

A
/
T
/
S
2
TABLE 4 (continued)
S
.

A
f
r
.

J
.

E
n
o
l
.

V
i
t
i
c
.
,
V
o
l
.

2
7
,
N
o
.

1
,
2
0
0
6
2
5
T
h
e

R
o
l
e

a
n
d

U
s
e

o
f

N
o
n
-
S
a
c
c
h
a
r
o
m
y
c
e
s

Y
e
a
s
t
s

i
n

W
i
n
e

P
r
o
d
u
c
t
i
o
n
Yeast species
[Synonym]
1
Country
or region
Isolation material
(must variety)
F
o
r
m

i
s
o
-
l
a
t
e
d

A
/
T
/
S
2
TABLE 4 (continued)
Kloeckera africana / Hanseniaspora vineae A Bordeaux Red grape variety Not given Peynaud & Domercq, 1959
A Italy Variety not given Not given Castelli, 1955
Kloeckera apiculata / Hanseniaspora uvarum A Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
A Alicante Monastrell (red) Dilutions plated on malt extract agar Querol et al., 1990
T Catalonia Macabeo (white) and Grenache (red) Dilution and plating on YPD Torija et al., 2001
T Catalonia Garnatxa Dilutions plated on malt extract agar Torija et al., 2001
A Majorca Chenin blanc & Prensal blanc Dilutions plated onto YM and lysine agar Mora & Mulet, 1991
A Australia Riesling, Semillon (white), Malbec and Spread inoculation on malt extract and lysine agar Heard & Fleet, 1985
Hermitage (red)
T Bordeaux Semillon Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
A Bordeaux Merlot Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
A Bordeaux Red & white varieties Not given Peynaud & Domercq, 1959
T Italy Nebbiola (red) Dilutions plated on Phytone yeast extract agar Schtz & Gafner, 1993
T Germany Pinot noir Dilutions plated on yeast extract agar Schtz & Gafner, 1993
Bordeaux Red varieties Not given Peynaud & Domercq, 1959
T Switzerland Pinot noir Dilutions plated on Phytone yeast extract agar Schtz & Gafner, 1993
T Switzerland Pinot noir Details not given Lorenzini, 1999
A Italy Different varieties Details not given Castelli, 1955
A Argentina Malbec Plated on malt extract agar Combina et al., 2005
Kloeckera apis / A Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Hanseniaspora guilliermondii
Kloeckera corticis / T Italy Variety not given Details not given Castelli, 1955
Hanseniaspora osmophila [K. magna]
Kloeckera javanica / Hanseniaspora S Bordeaux Red variety Not given Peynaud & Domercq, 1959
occidentalis [Kloeckera jensenii]
A Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
A Alicante Monastrell (red) Dilutions plated on malt extract agar Querol et al., 1990
T North Carolina Muscadine (Vitis rotundifolia) Direct plating on potato dextrose agar Parish & Carroll, 1985
Kluyveromyces thermotolerans Catalonia Macabeo (white) & Grenache (red) Dilution and plating on YPD Torija et al., 2001
Tenerife White Listan Random harvesting; further details not given De Cos et al., 1999
Pichia farinosa Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Pichia kluyveri Bordeaux Merlot Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
Pichia terricola / Issatchenkia terricola A Bordeaux Merlot Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
Rhodotorula sp. Bordeaux Merlot Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
S
.

A
f
r
.

J
.

E
n
o
l
.

V
i
t
i
c
.
,
V
o
l
.

2
7
,
N
o
.

1
,
2
0
0
6
2
6
T
h
e

R
o
l
e

a
n
d

U
s
e

o
f

N
o
n
-
S
a
c
c
h
a
r
o
m
y
c
e
s

Y
e
a
s
t
s

i
n

W
i
n
e

P
r
o
d
u
c
t
i
o
n
Yeast species
[Synonym]
1
Country
or region
Isolation material
(must variety)
F
o
r
m

i
s
o
-
l
a
t
e
d

A
/
T
/
S
2
TABLE 4 (continued)
Rhodotorula glutinis Bordeaux Merlot Dilutions plated on malt extract agar and grape juice agar Fleet et al., 1984
Tenerife White Listan Random harvesting; further details not given De Cos et al., 1999
Spain White and red (traditional varieties) Samples plated on Sabouraud dextrose agar Rementeria et al., 2003
Rhodotorula graminis Bordeaux Semillon Dilutions plated on malt extract agar & grape juice agar Fleet et al., 1984
Rhodotorula minuta Alicante Monastrell (red) Dilutions plated on malt extract agar Querol et al., 1990
Rhodotorula mucilaginosa Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Saccharomycodes sp. Spain Ribeiro Dilution and plating on YPD Cansado et al., 1989
[Saccharomyces ludwigii] S Tenerife White Listan Random harvesting; no further details. De Cos et al., 1999
Schizosaccharomyces spp. Catalonia Macabeo (white) and Grenache (red) Dilution and plating on YPD Torija et al., 2001
Torulaspora sp. Spain Ribeiro Dilution and plating on YPD Cansado et al., 1989
Bordeaux Red & white varieties Not given Peynaud & Domercq, 1959
Torulaspora globosa Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Torulopsis famata Bordeaux Red variety Not given Peynaud & Domercq, 1959
Zygosaccharomyces bailii Spain White and red varieties Samples of must diluted and plated Regueiro et al., 1993
on agar malt and Sabouraud plates
Zygosaccharomyces florentinus Spain Abarino, Godello (white) and Mencia (red) Dilution plated on juice-agar medium Longo et al., 1991
Zygosaccharomyces rouxii Argentina Malbec Plated on malt extract agar Combina et al., 2005
1
Where applicable, the anamorph and teleomorph designations are given, and also the synonym or alternate name used in older literature.
Yeast nomenclature given according to Kurtzman & Fell (1998).
2
A = anamorph; T = teleomorph; S = synonym.
27
The high numbers and sustained presence of non-
Saccharomyces yeasts in modern wine fermentations have result-
ed in wine microbiologists revisiting the role of these yeasts in
wine fermentation. Spontaneously fermented wines, although
carrying a higher risk of spoilage, are generally regarded as hav-
ing improved complexity, mouth-feel (texture) and integration of
flavours relative to inoculated wines (Heard & Fleet, 1985;
Bisson & Kunkee, 1991; Gil et al., 1996; Lema et al., 1996;
Grbin, 1999; Soden et al., 2000). This is due to specific metabol-
ic end products.
Documented cases include the study by Lema et al. (1996) on
Albario wine aroma components. These authors concluded that
the predominance of inoculated S. cerevisiae, along with a
notable growth rate of indigenous non-Saccharomyces yeasts dur-
ing the first days of the wine fermentation, contributed signifi-
cantly to the desirable aromatic properties of the wines. Herraiz
et al. (1990) and Gil et al. (1996) also reported that wines pro-
duced by pure and mixed cultures of S. cerevisiae and apiculate
yeasts (K. apiculata and H. uvarum) differ regarding their aro-
matic compounds. The low frequency of Kloeckera spp. during
fermentation has also been suggested as a reason for the lack of
aroma complexity of Folle blanche wines in the Basque region in
Spain (Rementeria et al., 2003).
The range of flavour compounds produced by different yeasts
is well documented (Castor, 1954; Suomalainen & Lehtonen,
1979; Soles et al., 1982; Nyknen, 1986; Rauhut, 1993; Romano
& Suzzi, 1993a; Lambrechts & Pretorius, 2000; Rojas et al.,
2003; Romano et al., 2003; Moreira et al., 2005; Swiegers &
Pretorius, 2005; Swiegers et al., 2005). The metabolic products
resulting from non-Saccharomyces growth include glycerol,
acetaldehyde, acetic acid, succinic acid, higher alcohols and fatty
acid esters (Fleet et al., 1984; Bisson & Kunkee, 1991; Jackson,
1994; Boulton et al., 1996; Lonvaud-Funel, 1996; Heard, 1999;
Zohre & Erten, 2002; Clemente-Jimenez et al., 2004).
Glycerol, after ethanol, is the next major yeast metabolite pro-
duced during wine fermentation and is important in yeast metabo-
lism for regulating the redox potential in the cell (Scanes et al.,
1998; Prior et al., 2000). Glycerol contributes to smoothness
(mouth-feel), sweetness and complexity in wines (Ciani &
Maccarelli, 1998), but the grape variety and wine style will deter-
mine the extent to which glycerol impacts on these properties. It
appears that the quality of Chardonnay, Sauvignon blanc and
Chenin blanc is not improved by increased glycerol concentrations
(Nieuwoudt et al., 2002). However, as all wine sensory profiles are
unique, some wines might benefit from increased glycerol levels.
Spontaneously fermented wines have higher glycerol levels,
indicating a possible contribution by non-Saccharomyces yeasts
(Romano et al., 1997; Henick-Kling et al., 1998). It is known that
C. stellata produces elevated glycerol concentrations of between
10 and 14 g/L (Ciani & Picciotti, 1995; Ciani & Ferraro, 1998),
compared with 4 to 10.4 g/L by S. cerevisiae (Radler & Schtz,
1982; Ciani & Maccarelli, 1998; Prior et al., 2000).
Unfortunately, increased glycerol production is usually linked to
increased acetic acid production (Prior et al., 2000), which can be
detrimental to wine quality. This makes the growth of C. stellata
during fermentation problematic, unless a balance can be
achieved between glycerol and acetic acid production, which will
not detract from wine quality.
Apiculate yeasts (K. apiculata and Hanseniaspora guillier-
mondii) have also been implicated in glycerol production. These
yeasts can generally be divided into two groups, i.e. high-glycerol
(3 g/L), low ethanol (0.9%
v
/v) producers, and low- glycerol
(1 g/L), high-ethanol (2.4%
v
/v) producers (Romano et al., 1997).
Apiculate yeasts are also known as high producers of acetic acid,
making them undesirable for wine production (Ciani & Picciotti,
1995). However, it has been reported that large strain variability
exists and that not all strains of Kloeckera spp. form high levels
of acetic acid (Romano et al., 1992). Some strains form less than
1 g/L and are comparable to S. cerevisiae. If these strains also
form higher levels of glycerol, their use can benefit a wine for
which higher glycerol levels are needed.
The primary flavour of wine is derived from the grapes.
However, secondary flavours are derived from ester formation by
yeasts during wine fermentation (Nyknen, 1986; Lambrechts &
Pretorius, 2000). Over 160 esters have been distinguished in wine
(Jackson, 2000). These esters can have a positive effect on wine
quality, especially in wine from varieties with neutral flavours
that are consumed shortly after production, e.g. some Chenin
blanc wines (Lambrechts & Pretorius, 2000). Non-Saccha-
romyces yeasts isolated from South African musts during 1961
could be divided into two groups, viz. neutral yeasts (producing
little or no flavour compounds) and flavour producing species
(both desired and undesired) (Van Zyl et al., 1963). Flavour pro-
ducing yeasts included P. anomala (Hansenula anomala) and
K. apiculata. C. pulcherrima is also known to be a high producer
of esters (Bisson & Kunkee, 1991; Clemente-Jimenez et al.,
2004). However, C. pulcherrima has an antagonistic effect on
several yeasts, including S. cerevisiae (Panon, 1997; Nguyen &
Panon, 1998). When aerobic growth of C. pulcherrima cultures
was followed by inoculation with S. cerevisiae, delays in fermen-
tation occurred. This was due to a killer effect, but it was not the
same as the classical S. cerevisiae killer phenomenon; it was
linked to pulcherrimin pigment produced by C. pulcherrima.
Contrary reports (Jolly et al., 2003b, 2003c) indicate that when
using a C. pulcherrima isolate for wine production there was no
effect on fermentation. This contradiction might be due to differ-
ent distinct biotypes within the C. pulcherrima species (Pallmann
et al., 2001), but this still needs to be investigated further.
Other yeasts that can play a role in wine production are those
of the genus Brettanomyces (Dekkera). Different Brettanomyces
strains can develop flavours ranging from pleasantly fruity and
toffee, to volatile and unpleasant (Eschenbruch & Wong, 1993).
The mousy and medicinal-like character in wine (known as
Brett), is linked to the growth of Brettanomyces spp. and is due
to the formation of tetrahydropyridines and 4-ethyl phenol,
respectively (Grbin et al., 1995; Grbin & Henschke, 2000; Arvik
& Henick-Kling, 2002). This Brett character is considered to be
an off-flavour. However, anecdotal evidence indicates that the
Brett character, at low levels in a red wine with an overall com-
plex aroma, can be a positive addition.
Some non-Saccharomyces yeasts, e.g. T. delbrueckii and
C. stellata are able to form succinic acid (Ciani & Maccarelli,
1998; Ferraro et al., 2000). This correlates with high ethanol pro-
duction and ethanol tolerance. Succinic acid production could
positively influence the analytical profile of wines by contribut-
ing to the total acidity in wines with insufficient acidity. However,
28
succinic acid has a salty/bitter-acid taste (Amerine at al., 1972)
and excessive levels will negatively influence wine quality.
Different non-Saccharomyces yeasts produce different levels of
higher alcohols (n-propanol, isobutanol, isoamyl alcohol, active
amyl alcohol) (Romano et al., 1992; Lambrechts & Pretorius,
2000). This is important during wine production, as high concen-
trations of higher alcohols are generally not desired, but lower
values can add to wine complexity (Romano & Suzzi, 1993b).
Non-Saccharomyces yeasts often form lower levels of these alco-
hols than S. cerevisiae, but there is great strain variability
(Romano et al., 1992, 1993; Zironi et al., 1993).
Other non-Saccharomyces metabolites can act as intermediates
in aroma metabolic pathways. Romano et al. (1993) showed that
H. guilliermondii and K. apiculata strains produced 50.3 to 258.1
mg/L and 55.8 to 187.4 mg/L acetoin, respectively, in grape must.
Acetoin is considered a relatively odourless compound in wine,
with a threshold value of approximately 150 mg/L (Romano &
Suzzi, 1996). However, diacetyl and 2,3-butanediol (potentially
off-flavours in wine) can be derived from acetoin by chemical
oxidation and yeast-mediated reduction, respectively. This indi-
cates that acetoin can play a role in off-flavour formation in
wines. High concentrations of acetoin produced by non-
Saccharomyces yeasts can also be utilised by S. cerevisiae. This
was shown by Zironi et al. (1993) in their chemical analysis
results of wines fermented from pure cultures, compared to
mixed and sequential culture fermentations. They could not, how-
ever, confirm what metabolites were formed from acetoin by
S. cerevisiae.
These, and other compounds not discussed in this article (e.g.
volatile fatty acids, carbonyl and sulphur compounds), are known
to play a role in the sensory quality of wine (Nyknen, 1986;
Lambrechts & Pretorius, 2000; Moreira et al., 2005). However, as
stated by Guth (1997), there are over 680 documented com-
pounds in wine and a large number of these can, depending on
their concentrations, contribute either positively or negatively to
wine aroma and flavour. It is not known how these compounds
relate to the metabolism of the different yeast species found in
fermentation.
Certain flavour and aroma compounds are present in grapes as
glycosidic precursors with no sensory properties (Todd, 1995;
Pretorius, 2003). These compounds may be hydrolysed by the
enzyme -glucosidase to form free volatiles that can improve the
flavour and aroma of wine, but this enzyme is not encoded by the
S. cerevisiae genome (Ubeda-Iranzo et al., 1998; Van Rensburg et
al., 2005). However, certain non-Saccharomyces yeasts belong-
ing to the genera Debaryomyces, Hansenula, Candida, Pichia
and Kloeckera possess various degrees of -glucosidase activity
(Rosi et al., 1994; Todd, 1995; Spagna et al., 2002; Fernndez-
Gonzles et al., 2003; Rodriguez et al., 2004) and can play a role
in releasing volatile compounds from non-volatile precursors. An
intracellular -glucosidase has also been isolated and purified
from Debaryomyces hansenii. This enzyme, which is not inhibit-
ed by glucose and ethanol, was used during fermentation of
Muscat grape juice, resulting in an increase in concentration of
monoterpenols in the wine (Yanai & Sato, 1999).
As already mentioned, ester formation by yeast plays an impor-
tant role in secondary flavours. However, the net accumulation of
esters in wine is determined by the balance between the yeasts
ester-synthesising enzymes and esterases (responsible for cleav-
age and, in some cases, formation of ester bonds) (Swiegers &
Pretorius, 2005). Although extracellular esterases are known to
occur in S. cerevisiae (Ubeda-Iranzo et al., 1998), the situation
for non-Saccharomyces needs further investigation.
Other non-Saccharomyces extracellular enzymatic activity (e.g.
proteolytic, polygalacturonase) might also be of use in winemak-
ing. For example, proteolytic activity of some non-Saccharomyces
yeasts could lead to a reduction in protein levels, with an accom-
panying increase in protein stability. However, Dizy & Bisson
(2000) reported to the contrary, namely that increased yeast prote-
olytic activity did not lead to a reduction in haze formation. Some
extracellular enzymes produced by non-Saccharomyces species
are shown in Table 5. Species found to produce the greatest num-
ber of extracellular enzymes are C. stellata, H. uvarum/K. apicu-
lata and M. pulcherrima/C. pulcherrima.
New research by Carrau et al. (2005) on the formation of aroma
compounds by yeasts suggests that S. cerevisiae can synthesise
monoterpenes (floral aroma in wine), compounds previously
thought to be solely derived from grapes. They have also pro-
posed a new metabolic pathway (MCC pathway). It is unknown
whether any non-Saccharomyces yeasts have these capabilities.
However, considering the large variety of non-Saccharomyces
yeast species found in grape must, it is likely that there will be
some.
FACTORS AFFECTING GROWTH OF NON-SACCHARO-
MYCES YEASTS DURING WINE FERMENTATION
The contribution by non-Saccharomyces yeasts to wine flavour
will depend on the concentration of metabolites formed.
Therefore, any factors affecting the growth rate of individual non-
Saccharomyces species will determine the extent of their contri-
bution to flavour development (Heard, 1999). These factors
include intrinsic components of grape juice (pH, sugar concen-
tration and clarity), processing methods (method of clarification,
use of preservatives and/or SO2), fermentation conditions (tem-
perature, oxygen content/aeration) and the effect of S. cerevisiae
yeast, e.g. amount of ethanol formed.
Non-Saccharomyces yeasts have poor tolerance to low oxygen
availability, especially compared with S. cerevisiae (Hansen et
al., 2001). The removal of oxygen by vigorously fermenting
S. cerevisiae can contribute to an early death of some non-
Saccharomyces yeasts.
For some non-Saccharomyces yeasts, i.e. C. stellata, C. col-
liculosa and C. pulcherrima, it has been shown that the pH of the
medium or must does not have a large effect on growth rate (Gao
& Fleet, 1988; Heard & Fleet, 1988), however, it has also been
reported that higher pH increases the fermentation ability of
C. pulcherrima (Jolly et al., 2003c). The effect of pH on other
non-Saccharomyces yeasts is not well defined. An increase in
sugar concentration, however, does have an effect on non-
Saccharomyces yeasts due to their generally poor osmotolerance
(Heard, 1999).
Temperature and SO2 are possibly the two factors that have the
greatest effect on non-Saccharomyces yeasts. Some non-
Saccharomyces yeasts are inhibited at temperatures above 25C
(Sharf & Margalith, 1983; Fleet, 1990; Boulton et al., 1996).
29
Therefore, reducing the temperature can lead to greater contribu-
tion by non-Saccharomyces species. K. apiculata, for example, is
able to ferment better at 10C than at 25C (Heard & Fleet, 1988;
Bilbao et al., 1997). The ethanol tolerance of some non-
Saccharomyces yeasts, e.g. C. stellata and K. apiculata, is also
improved at lower temperatures (e.g. 10C compared with 25C)
(Gao & Fleet, 1988). Other non-Saccharomyces yeasts, e.g.
H. guilliermondii already have an ethanol tolerance very similar
to S. cerevisiae, however, this is influenced by prior growth con-
ditions (Pina et al., 2004). Reducing the fermentation tempera-
ture, with its synergistic effect on ethanol tolerance, will therefore
lead to an increased presence of non-Saccharomyces yeasts. This
change in the ecology of the fermentation will also result in a
change in the concentration of aroma and flavour metabolites
formed by the non-Saccharomyces yeasts, with a consequential
impact on wine quality.
While it has generally been accepted that SO2 added to wine
suppresses non-Saccharomyces yeasts, the work of Henick-Kling
et al. (1998), Constant et al. (1998), Egli et al. (1998) and
Rementeria et al. (2003) challenges this belief. Low sulphur addi-
tion (20 mg/L) does not suppress non-Saccharomyces yeasts,
higher levels (50 mg/L) do suppress some, but others, e.g. C. guil-
liermondii and Zygosaccharomyces spp., can survive. The effec-
tiveness of SO2 also depends on the type of must and the number
of non-Saccharomyces yeasts present in the first place. Results of
Fleet (1990) and Granchi et al. (1998) support this. They found
that SO2 in the range 50 to 100 mg/L did not prevent growth of
non-Saccharomyces yeasts in red wine fermentations, but these
concentrations are normally effective in white wine fermenta-
tions. Suppression of sensitive yeasts by SO2 allows more toler-
ant yeasts, present in lower numbers, to proliferate. The judicious
use of SO2 can therefore alter the non-Saccharomyces population
profile in some instances. In other instances, the addition of SO2
will decrease some populations, but not the species diversity
(Rementeria et al., 2003).
Other factors that can impact on the presence and growth of
non-Saccharomyces yeasts are yeast-yeast and yeast-bacteria
interactions, including neutralism, commensalism, mutualism/
synergism, amensalism or antagonism and competition (Boddy &
Wimpenny, 1992; Boulton et al., 1996; Henick-Kling et al., 1998;
Fleet, 2003). The utilisation of specific nutrients, e.g. amino acids
and vitamins, by one species may make the environment less
favourable to other species. In addition, metabolites produced,
such as ethanol, inhibitory peptides, proteins and glyco-proteins,
can inhibit or destroy other species by lysis of their cell walls.
Specifically, zymocidal strains of S. cerevisiae (killer yeasts) can
affect other species. Other metabolites can, in turn, lead to
enhanced growth. Autolysis, with the subsequent release of cellu-
Extracellular enzyme activity
Species
(teleomorph / anamorph)
TABLE 5
Extracellular hydrolytic enzymes found in non-Saccharomyces yeasts.
Zygoascus hellenicus / Candida hellenica + + + S
Brettanomyces clausenii + F
Kluyveromyces thermotolerans + F
Pichia fermentans / Candida lambica + + S
-2
/ Candida oleophila + + + S
Issatchenkia occidentalis / Candida sorbosa + + S
-2
/ Candida stellata + + + + + + + + + C, F, S
Pichia membranifaciens / Candida valida + + + + F, S
Debaryomyces hansenii / Candida famata + + + R
4
, S, B
Hanseniaspora uvarum / Kloeckera apiculata + + + + + + + + + R
4
, C, S, Ro
4
Issatchenkia orientalis / Candida krusei + C
Metschnikowia pulcherrima / Candida pulcherrima + + + + + + + + + + C, F, S, Ro
Pichia anomala / Candida pelliculosa + + + R
4
, C, F, S, Sp
Torulaspora delbrueckii / Candida colliculosa + C
Pichia farinosa /
-3
+ S
Debaryomyces castellii /
-3
+ R
4
Debaryomyces polymorphus /
-3
+ R
4
Pichia kluyveri /
-3
+ S
Pichia guilliermondii / Candida guilliermondii + Ro
4
1
R = Rosi et al., 1994; C = Charoenchai, et al., 1997; F = Fernndez et al., 2000; S = Strauss et al., 2001; Sp = Spagna et al., 2002 ; Ro = Rodriguez et al., 2004, and
B = Bolumar et al., 2005.
2
No teleomorph.
3
No anamorph.
4
Only screened for -glucosidase activity.
P
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t
i
n
a
s
e
P
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e
a
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e
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n
a
s
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-
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o
s
i
d
a
s
e
C
e
l
l
u
l
a
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X
y
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a
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a
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30
lar material, can also encourage yeast growth (Charpentier et al.,
1986; Fleet, 2003).
Growth of non-Saccharomyces yeasts can also be limited by the
S. cerevisiae starter culture (Constant et al., 1998; Henick-Kling
et al., 1998). High concentrations of S. cerevisiae appear to inhib-
it some non-Saccharomyces yeasts by means of a cell-cell mediat-
ed mechanism (Nissen et al., 2003). Subsequently, different starter
cultures will have different effects on the non-Saccharomyces pop-
ulations. Therefore, by controlling the parameters of inoculum, i.e.
S. cerevisiae strain and inoculum concentration, the non-
Saccharomyces contribution can also be controlled. This is illus-
trated by the experiments of Egli et al. (1998). They compared
three types of fermentation, i.e. fermentations with indigenous
microflora (spontaneous), a vigorous yeast starter (S. cerevisiae
strain EC1118) and a slowly fermenting yeast starter (S. cerevisi-
ae strain Assmanshausen). The sensory profiles of the resultant
wines differed from each other. The faster growing EC1118 strain
limited the growth of the non-Saccharomyces component more
strongly than the slow growing Assmanshausen. Growth could be
further suppressed by the addition of SO2.
It has also been suggested that the use of fungicides and pesti-
cides results in a reduced yeast presence on grapes (Parish &
Caroll, 1985; Regueiro et al., 1993; Guerra et al., 1999). It has
been shown that yeast populations are higher when no agrochem-
icals have been applied for some time (Renouf et al., 2005).
Higher levels of non-Saccharomyces yeasts can impact on the
resultant fermentation and wine quality. In contrast, Cabras et al.
(1999) showed that certain pesticides could stimulate yeasts
(especially K. apiculata) to produce more ethanol. This increased
vigour would, by implication, also extend to other metabolites. As
pesticides are used to a greater or lesser extent in all vineyards
(with a few possible exceptions where natural processes are fol-
lowed), there might be instances where certain pesticides can
stimulate non-Saccharomyces growth on the grapes. The resultant
higher levels of such yeasts might also have a knock-on effect in
the subsequent wine fermentation.
Non-Saccharomyces populations might also be affected by
quorum sensing, the mechanism whereby microbial cells can
communicate with each other and cause a population to follow a
specific growth pattern (Bisson, 1999; Fleet, 2003; Parsek &
Greenberg, 2005). Bicarbonate, ammonia and farnesol have been
suggested as cell communication molecules for yeast. The effect
of quorum sensing and its significance to wine production is not
known.
THE USE OF NON-SACCHAROMYCES YEASTS IN WINE
PRODUCTION
A number of authors have reported that the use of selected and
cultured non-Saccharomyces yeasts, including Candida,
Kloeckera, Brettanomyces and Zygosaccharomyces species, is
beneficial in wine production. As these yeasts are all poor fer-
menters, S. cerevisiae (either indigenous or inoculated) is always
needed to complete wine fermentation. Typically, non-
Saccharomyces yeasts have been used in sequential fermentation
where these yeasts are allowed to grow or ferment for between
one hour and fifteen days before inoculation with S. cerevisiae
(Herraiz et al., 1990; Zironi et al., 1993; Ciani & Ferraro, 1998;
Ferraro et al., 2000; Jolly et al., 2003b; 2003c). Unfortunately,
some authors only report on experiments conducted on laborato-
ry-scale, utilising small volumes of grape juice. These results are
not necessarily the same as what could be expected in larger com-
mercial fermentations. Factors such as small amounts of air
which can enter small-volume fermentations during e.g. sam-
pling, and the rapid sedimentation of yeast which reduces fer-
mentation rate, can affect the final results (Henschke, 1990).
Candida stellata
C. stellata is known as a high glycerol producer and Ciani &
Picciotti (1995) suggested that it be used as a starter culture to
increase glycerol levels in wine. Values of up to 11.76 g/L have
been reported (Ciani & Maccarelli, 1998), which is higher than
the sensory threshold level for glycerol sweetness, i.e. 5.2 g/L
(Noble & Bursick, 1984). Glycerol is also thought to contribute
to the mouth-feel and complexity of wine flavour, at lower levels
(Scanes et al., 1998; Prior et al., 2000). This yeast is therefore a
prime candidate for investigating a positive non-Saccharomyces
contribution to wine quality.
Ciani & Ferraro (1998) used a C. stellata strain (strain 3827,
Industrial Yeast Collection, University of Perugia) to improve the
analytical profile of small-scale (500 mL) Pinot grigio wines dur-
ing batch fermentations. The Pinot grigio grapes were harvested
at 18.5B and sweetened to 27B with sucrose. Immobilised
C. stellata cells at concentrations of 1 x 10
9
cells/mL were used
in three fermentation types: a simultaneous inoculation with
S. cerevisiae; a sequential fermentation where S. cerevisiae was
added three days after C. stellata; and a substituted fermentation
where C. stellata was replaced with S. cerevisiae after three days.
The S. cerevisiae inoculum was 1 x 10
6
cells/mL for all treat-
ments. Results showed that in comparison to the S. cerevisiae
control fermentations, the combined fermentations occurred more
rapidly with increased glycerol content. This was accompanied
by a decrease in acetic acid and higher alcohols and an increase
in succinic acid. Other by-products were similar to those found in
the S. cerevisiae control fermentation. Despite the high initial
sugar content, all of the sugar was consumed due to the comple-
mentary utilisation of fructose and glucose by the C. stellata
(fructophilic yeast) and S. cerevisiae (glucophilic yeast). It was
concluded that of all three treatments, the sequential fermentation
was the best combination for improving the analytical profile of
the wines. It was further noted that acetoin produced by C. stel-
lata was utilised by S. cerevisiae to form 2,3-butanediol that can
lead to off-flavour in wine. As no sensory analyses were carried
out it is not clear what impact this had on the flavour of the wine.
In a subsequent study, Ferraro et al. (2000) confirmed the above
findings in the pilot-scale (100 L) production of wine. They used
Trebbiano Toscano grape juice at 19B, pH 2.94 and 11.52 mg/L
free SO2. The inoculum of immobilised C. stellata
(5 x 10
8
cells/mL) was followed after three days by an inoculum
of S. cerevisiae (5 x 10
6
cells/mL). High glycerol production was
evident on the fourth day of fermentation, while the alcohol was
still lower than 5%. Thereafter, the glycerol production was slow-
er and the ethanol concentration increased due to metabolism by
S. cerevisiae. The final wine had a 70% higher glycerol concen-
tration than the control (S. cerevisiae only), while the ethanol con-
centration was 10.6% compared with the 12.24% of the control.
The acetic acid was 0.05 g/L lower than the control fermentation.
31
Apart from the increase in glycerol, the reduction in alcohol as
reported above could be beneficial for the production of wines
from grapes with high sugar content (as often found in grapes
from warm regions). Reduction in acetic acid concentration will
always be beneficial. However, while the analytical profile of the
wine was improved, a shortcoming of the work of Ferraro et al.
(2000) is that they did not report on the sensory profile of the
wine. During the three-day lag before inoculation with S. cere-
visiae, oxidation of the must might have occurred. However,
spontaneous fermentation would have started and this might have
offset the oxidation risk.
In another investigation Soden et al. (1998; 2000) used C. stel-
lata in combination with S. cerevisiae for the production of
Chardonnay wines. They used two different inoculation proto-
cols, viz. co-inoculation and sequential inoculation, starting with
the C. stellata, and these were compared with the two yeasts in
separate monoculture ferments. The yeasts were inoculated at a
concentration of 5 x 10
6
cells/mL, except in the co-inoculated fer-
mentation where the S. cerevisiae was inoculated at a lower cell
count (5 x 10
5
cells/mL). In the sequential fermentation, S. cere-
visiae was inoculated 15 days after C. stellata. At this point the
C. stellata had depleted the fructose, but not the glucose, and had
stopped fermenting. All the wines except the C. stellata mono-
culture fermented dry.
The wines underwent descriptive sensory analyses and the ref-
erence S. cerevisiae wine was judged to have tropical fruit, flo-
ral, lime and banana aromas of a typical Chardonnay. The
monoculture C. stellata wine had significantly more apricot,
honey and sauerkraut aromas and significantly less lime,
tropical fruit, banana and floral aromas. The co-inoculated
wine had aromas similar to wine resulting from use of the S. cere-
visiae monoculture, but was scored lower for floral and
banana. The sequential fermentations produced a wine signifi-
cantly different from the S. cerevisiae reference wine regarding
banana, floral and lime aromas, but the wine was similar in
the honey, apricot and sauerkraut aromas attributed to the
C. stellata yeast. The wine also had a high ethyl acetate aroma,
had the highest concentration of glycerol and succinic acid, and a
lower concentration of ethanol. On the negative side, sauerkraut
and ethyl acetate nuances could be considered to detract from
wine quality as they are listed under microbiological and oxi-
dised according to wine evaluation terminology (Noble et al.,
1987). The long time lapse of 15 days before the inoculation with
S. cerevisiae in the sequential fermentation would have con-
tributed to the oxidised aroma of the wine. A shorter delay
before inoculation with S. cerevisiae could have given a better
wine. Soden et al. (2000) concluded that, with selection of the
appropriate strains and the establishment of effective inoculation
protocols, greater flavour diversity and complexity could be
obtained in wine during commercial winemaking.
In another study where C. stellata and S. cerevisiae were used
in sequential fermentations for the production of small-scale
Chardonnay wines, the time lapse between inoculations of the
two species was only one hour (Jolly et al., 2003b). No increases
in glycerol levels were noted, as was the case when the C. stella-
ta isolate was used on its own in laboratory-scale fermentations.
For the small-scale Chardonnay wine, the total esters was signif-
icantly higher for the C. stellata sequential wine in comparison to
the S. cerevisiae only reference wine. However, during a compar-
ative sensory evaluation the reference wine was preferred. No
descriptive sensory analysis was performed.
Candida pulcherrima
C. pulcherrima, which produces high concentrations of esters
(Bisson & Kunkee, 1991), especially the pear-associated ester
ethyl caprylate (Lambrechts & Pretorius, 2000; Clemente-Jimenez
et al., 2004), can occur in high numbers in grape must (Schtz &
Gafner, 1993; Jolly et al., 2003a). Furthermore, it was shown by
Zohre & Erten (2002) that production of undesirable volatile com-
pounds did not occur during mixed culture fermentations of this
yeast and S. cerevisiae. C. pulcherrima might therefore make a
positive sensory contribution to wine. In another study, a random-
ly selected C. pulcherrima isolate was used in sequential fermen-
tations with S. cerevisiae, for small-scale production of
Chardonnay, Sauvignon blanc and Chenin blanc wines (Jolly et
al., 2003b). A sensory evaluation of the wines showed that
Sauvignon blanc and Chenin blanc were better than a reference
wine (S. cerevisiae only) five and 18 months after production. The
Chardonnay wine was judged to be of an inferior quality. It thus
appears that there are specific non-Saccharomyces/grape variety
combinations that lead to improved quality.
In a subsequent study, the effect of winemaking practices
(namely the use of diammonium phosphate [DAP] and SO2), fer-
mentation temperatures and must pH on the performance of
C. pulcherrima during fermentation was investigated (Jolly et al.,
2003c). It was reported that DAP addition, higher pH values and
increased temperatures all resulted in a slight increase in fermen-
tation ability. The fermentation ability was not affected by SO2
concentrations normally used in wine fermentation (0-30 mg/L).
Elevated levels (60 mg/L) of SO2 did have a negative effect, how-
ever, this is a much higher concentration than would normally be
used in a commercial fermentation. A Chenin blanc wine produc-
tion trial showed that a selected C. pulcherrima strain had a pos-
itive influence on wine quality (as measured by a sensory panel).
No difference in standard chemical analyses was noted and the
improvement was not linked to ester levels; the authors implicat-
ed other metabolites, but further chemical analyses and suitable
methodology were required to identify them.
Kloeckera and Hanseniaspora spp.
The apiculate yeasts K. apiculata and H. uvarum, the non-
Saccharomyces yeasts found in the highest numbers in grape
must, are in the best position to make a contribution to wine qual-
ity. These yeasts, with low fermentative power, are important in
the production of volatile compounds in wine, and the chemical
composition of wines made with Kloeckera spp./S. cerevisiae
combinations differ from reference wines (Herraiz et al., 1990;
Mateo et al., 1991; Zironi et al., 1993; Gil et al., 1996).
Caridi & Ramondino (1999) evaluated a range of 20
Hanseniaspora spp. (Kloeckera spp.) of oenological origin for
their ability to ferment a must (17.9B; pH 3.81). They found that
the ethanol produced ranged from 5.02 to 8.72%, in comparison
to the 11.17% of the control (S. cerevisiae), but volatile acidity
was higher (0.75g/L to 2.25g/L) than the control (0.65g/L). These
results indicate that although some strains of Hanseniaspora are
able to produce higher levels of ethanol than other strains, the
high levels of volatile acidity would be detrimental to the senso-
32
ry characteristics of the wine. However, Romano et al. (1992) and
Ciani & Maccarelli (1998) showed that not all Kloeckera strains
formed high levels of volatile acidity and that some were similar
to S. cerevisiae in this regard. The production of other secondary
metabolites, i.e. glycerol, acetaldehyde, ethyl acetate and hydro-
gen sulphide, also differed between strains (Romano et al., 1997).
Selected strains of apiculate yeasts might therefore favour aroma
and flavour enhancement in wines.
Most authors working with these yeasts only utilised Kloeckera
spp. in small-scale laboratory trials and subsequently only
analysed the wines chemically. Their approaches to the use of
Kloeckera also differed. Zironi et al. (1993), in their sequential
fermentations, allowed a Kloeckera/Hanseniaspora ferment to go
for six days before inoculating with S. cerevisiae, while Herraiz
et al. (1990) waited eight days. Differences in chemical analyses
of the wines were noted. Of more concern was that the initial
growth of Kloeckera had a retarding effect on the subsequent
growth of S. cerevisiae. This phenomenon could have further
implications as a cause for lagging or stuck fermentations.
Therefore, a cautionary approach would have to be taken when
considering using Kloeckera spp. in wine production. K. apicula-
ta has also been implicated in the formation of some biogenic
amines (Caruso et al., 2002).
Sensory evaluations on wines produced by Kloeckera spp. were
carried out by Owuama & Saunders (1990). They used K. apicula-
ta/S. cerevisiae combinations for the fermentation of cashew apple
juice (25B). Inoculation of the two species was simultaneous and
9.3% ethanol was produced with 4.2% residual sugar. Sensory eval-
uation (colour, aroma and taste) of the wines showed that although
the Saccharomyces reference wine was the best, the product of the
combined fermentation was also acceptable for consumption.
A K. apiculata isolate was also used with S. cerevisiae for the
production of small-scale Chardonnay, Sauvignon blanc and
Chenin blanc wines (Jolly et al., 2003b). Inoculation of the
S. cerevisiae was one hour after that of the K. apiculata and ca.
13% ethanol was produced with less than 2 g/L residual sugar.
Sensory evaluation of the wines five and 18 months after produc-
tion showed that the Sauvignon blanc wine was preferred by the
judges. All the other wines were judged to be inferior to a refer-
ence wine produced by S. cerevisiae only.
The production of 2-phenyl-ethyl acetate by the apiculate yeast
H. guilliermondii has been investigated in laboratory fermentations
(Rojas et al., 2003). This acetate ester contributes to rose, honey,
fruity and flowery aroma nuances (Lambrechts & Pretorius,
2000; Swiegers & Pretorius, 2005; Swiegers et al., 2005), and is
formed to a greater or lesser extent by most yeasts. As part of the
fermentation bouquet, it can contribute to the overall flavour of a
young wine. However, the high level of ethyl acetate produced by
the strain investigated by Rojas et al. (2003) was a serious handicap.
Another role that has been suggested for Kloeckera and
Hanseniaspora spp. is that of the production of wine destined for
vinegar manufacture (Ciani & Picciotti, 1995). The high produc-
tion of acetoin and ethyl acetate will favourably influence the
quality of vinegar.
Zygosaccharomyces spp.
Zygosaccharomyces spp. are considered to be winery contami-
nants and are especially a problem in wineries producing sweet
and sparkling wines (Amerine & Cruess, 1960; Loureiro &
Malfeito-Ferreira, 2003). Notwithstanding, Zygosaccharomyces
spp. have been investigated by Romano & Suzzi (1993a) as pos-
itive contributors to wine fermentation. The species studied
included a Z. fermentati strain (strain F42), which produced low
levels of acetic acid, H2S and SO2 and had high fermentation
vigour. Another species, Z. bailii (strain F37), showed malic acid
degradation and generally low H2S production. In addition, both
species flocculated. The authors suggested that these characteris-
tics could benefit wine production during, for example, re-fer-
mentation of wine.
Most of the literature recognises Zygosaccharomyces yeasts as
spoilage organisms producing high quantities of acetic acid.
However, Romano & Suzzi (1993a) suggested that this acetic
acid production might be due to yeasts bearing a close resem-
blance to Zygosaccharomyces. In older literature, acetic acid-pro-
ducing yeasts were wrongly identified as Zygosaccharomyces
species.
Selected strains of Zygosaccharomyces spp. might be useful
especially for the production of alternate beverages. Z. bailii is
also, in contrast to many other non-Saccharomyces yeasts and
S. cerevisiae, fructophilic. This could be beneficial in grape musts
from riper grapes (high Balling), where the fructose concentra-
tion can exceed that of glucose at the start of fermentation
(Margalith, 1981).
Schizosaccharomyces spp.
Schizosaccharomyces spp. have been used for the production of
mango (Magnifera indida L.) wine without addition of S. cere-
visiae (Obisanya et al., 1987). Two strains of Schizosaccha-
romyces spp. isolated from palm wine were found to be suitable
for the production of sweet mango table wine with between 8 and
9% alcohol. Reference wines produced by S. cerevisiae were,
however, found to be superior in flavour and taste, while fer-
menting dry (approximately 4 g/L sugar).
Another characteristic of Schizosaccharomyces spp. is their
ability to degrade malic acid; it has been shown that high-density
cell suspensions of Schizosaccharomyces yeasts could degrade
95-99% of malic acid in a buffered assay system (Gao & Fleet,
1995). However, none of the yeasts could metabolise malic acid
fast enough to be of use in a reactor system for the treatment of
grape juice. Increasing the cell density had no improved effect.
In another trial, a Schizosaccharomyces malidevorans mutant
that could utilise malic acid more rapidly than the wild-type strain
was used for commercial-scale (1000 to 2500 L) deacidification
of grape juice (Thornton & Rodriguez, 1996). The varieties treat-
ed were Chardonnay, Semillon and Cabernet Sauvignon. The
temperature and SO2 levels were those normally applied in wine
production, i.e. 15-25C and 30 mg/L (free), respectively. No sen-
sory defects were noted in the finished wines, which were used
for blending, before being sold as varietal wines.
Torulaspora delbrueckii
T. delbrueckii (teleomorph of C. colliculosa), formerly classified
as Saccharomyces rosei, was previously suggested for vinifica-
tion of musts low in sugar and acid (Castelli, 1955). It was sub-
sequently used for the commercial production of red and ros
wines in Italy (Castelli, 1955). In more recent experiments carried
out by Moreno et al. (1991) it was found that T. delbrueckii in
33
pure culture produced lower levels of volatile acidity than the
S. cerevisiae strains tested. Furthermore, in mixed fermentations
(indigenous population plus pure cultures of T. delbrueckii or
S. cerevisiae) the volatile acidity increased with the ripeness of
the grapes, however, these increases were generally smaller for
the T. delbrueckii than the S. cerevisiae strains.
In another investigation, the anamorphic form of this yeast
(C. colliculosa) was used together with S. cerevisiae for the pro-
duction of small-scale Chardonnay, Sauvignon blanc and Chenin
blanc wines (Jolly et al., 2003b). The Sauvignon blanc and
Chenin blanc wines were both judged to be better than their
respective S. cerevisiae reference wines five and 18 months after
production. However, standard wine chemical and ester analyses
did not differ from the reference wines.
Brettanomyces spp.
The contribution of Brettanomyces spp. to wine aroma has been
likened to a Bordeaux-like character. Apart from the previously
mentioned negative aroma nuances imparted by these yeasts, pos-
itive aromas such as smoky, spicy and toffee are also cited
(Eschenbruch & Wong, 1993; Arvik & Henick-Kling, 2002).
Brettanomyces spp. have also been implicated in Belgian acidic
ale production where it is found in the final 20 to 24 month stage
in the fermentation casks (Martens et al., 1997). While the delib-
erate inoculation of Brettanomyces in must or wine for commer-
cial production has not been reported, some winemakers are
working with the indigenous populations of Brettanomyces found
in their cellars to make more complex wines, some of which are
highly regarded because of their aromas and flavours (Arvik &
Henick-Kling, 2002). This topic is being investigated by different
research groups (Grbin & Henschke, 2000; Arvik & Henick-
Kling, 2002) and a favourable Brettanomyces strain or protocol
for the beneficial use of Brettanomyces spp. for wine production
might still be found. However, the formation of mousy and
medicinal off-flavour compounds will have to be controlled.
Strain selection will also have to ensure that no biogenic amines
are produced (Caruso et al., 2002).
S. ludwigii is a lesser known, lemon-shaped yeast, typically with
a large cell size, frequently isolated from wine at the end of fer-
mentation or from wine in storage (Romano et al., 1999).
Secondary metabolites produced at high levels by this yeast
include isobutyl alcohol, acetoin and ethyl acetate. It is also
known to be highly resistant to SO2 and tolerant to ethanol. A
selected strain of S. ludwigii was used to ferment feijoa or pineap-
ple guava (Feijoa sellowiana) juice. The resultant beverage was
evaluated for aroma, flavour and taste by a consumer panel.
Despite the high levels of acetic acid, the beverage was described
as fresh with a fruity flavour, akin to apple and kiwi, and sim-
ilar to apple juice in taste, but with more acid. Romano et al.
(1999) concluded that this beverage had potential as a refreshing
summer drink.
Kluyveromyces thermotolerans
A commercial active dried yeast blend of K. thermotolerans and
S. cerevisiae (Viniflora
SYMPHONY.nsac) has recently been

released (Anon., 2004a). This combination has been developed
for the enhancement of aroma and flavour in white (Chardonnay,
Pinot blanc, Pinot gris and Riesling) and red (Cabernet
Sauvignon, Merlot, Shiraz and Pinot noir) grape varieties.
According to the product information sheet, the use of this yeast
in simultaneous inoculations can lead to the enhancement of flo-
ral and tropical fruit aromas, and more complex and rounded
flavours in white and red wine, respectively. Although the ratio of
the K. thermotolerans to S. cerevisiae is not specified, it appears
to be in the region of 1:30 (Jolly, unpublished data, 2005).
Other non-Saccharomyces spp.
Other non-Saccharomyces yeasts that have also been investigated
for their potential contribution to wine include Pichia and
Williopsis spp.
The production of 2-phenylethanol by Pichia fermentans has
been investigated under optimised culture conditions by Huang et
al. (2001). This alcohol, contributing to an aroma of rose petals, is
formed to a greater or lesser extent by most yeasts, and as part of
the fermentation bouquet can contribute to the overall flavour of
a young wine. It was found that the production of this compound
increased with an increase in biomass during the initial stage of fer-
mentation. A maximum concentration was reached after 16 hours.
The use of P. fermentans was also investigated by Clemente-
Jimenez et al. (2005) in microvinifications (250 mL). It was
found that mixed fermentations with S. cerevisiae resulted in an
increase in some aromatic compounds such as acetaldehyde,
ethyl acetate, 1-propanol, n-butanol, 1-hexanol, ethyl caprylate,
2,3-butanediol and glycerol.
Other Pichia spp., i.e. P. anomala, P. membranifaciens and
P. subpelliculosa, together with Williopsis saturnus have also
been suggested for the production of low-alcohol wines (ca.
3%
v
/v) in an aerated vessel (Erten & Camphill, 2001). In this
method stirring and agitation resulted in more yeast biomass
being formed and decreased ethanol production from fermentable
sugars. The wines, made by P. subpelliculosa and W. saturnus in
particular, were acceptable to a small tasting panel. This method
of low-alcohol wine production eliminates the need for costly and
complex post-production removal of alcohol from wines pro-
duced during normal fermentations. The higher levels of flavour
compounds, especially esters, produced by these aerobic yeasts
appeared to counteract the loss of flavour enhancing properties of
alcohol. The body of the wine normally attributed to ethanol
also appeared to have been replaced by the higher concentration
of esters.
The demand by the beverage industry for new and interesting
products and the well-developed flavour produced by aerobic
non-Saccharomyces yeasts makes this technology attractive.
However, problems envisioned are the need for non-standard fer-
mentation equipment and the reluctance of wine and beverage
producers to deliberately cultivate organisms normally consid-
ered as contaminants.
The early death of some non-Saccharomyces yeasts during fer-
mentation can also be a source of specific nutrients for S. cere-
visiae, enabling it to ferment optimally. These nutrients include
cellular constituents such as cell wall polysaccharides (manno-
proteins). For this method of nutrient supply to be effective, any
killer or other inhibitory effects by the non-Saccharomyces yeasts
against S. cerevisiae should be known (Herraiz et al., 1990;
Panon, 1997; Nguyen & Panon, 1998; Fleet, 2003) so that the
subsequent S. cerevisiae fermentation is not adversely affected.
34
Combinations of non-Saccharomyces yeasts
Eschenbruch & Wong (1993) inoculated a red grape variety
(Blauburger) with a combination of the non-Saccharomyces
yeasts Kloeckera sp. and Brettanomyces sp. This was followed
after 24 hours with a S. cerevisiae pure culture. These authors
reported that the wine developed an overall flavour and complex-
ity that they termed a Brettanomyces character. This character,
they maintained, was a direct result of the contribution of the
Kloeckera sp. and Brettanomyces sp. They reported further that a
lower cell concentration should be used as higher cell concentra-
tions (10
6
cells/mL) resulted in off-characters. Their control fer-
mentations showed an indigenous population of Kloeckera and
Candida yeasts, but the numbers of these declined and essential-
ly disappeared as the fermentation proceeded. No Brettanomyces
yeasts were detected. After inoculation of the control, the S. cere-
visiae yeast quickly became the predominant species. This was
also noted in the non-Saccharomyces inoculated fermentations.
The numbers of Candida sp. once again decreased as the fermen-
tation proceeded. However, the cell densities of the
Brettanomyces and Kloeckera (represented by the added yeasts
and indigenous yeasts already present) decreased only slightly
during the fermentation. These yeasts therefore could contribute
towards the fermentation metabolism, resulting in a contribution
to taste and flavour differences.
In another investigation with combinations of non-
Saccharomyces yeasts, T. delbrueckii was used in sequential fer-
mentations with K. apiculata. The K. apiculata was inoculated
first, followed three days later by T. delbrueckii, and finally
S. cerevisiae was inoculated after eight days. The wines produced
had volatile compositions different from the S. cerevisiae wines,
but were not subjected to sensory evaluation (Herraiz et al., 1990).
Another commercial active dried wine yeast culture (Viniflora
HARMONY.nsac) combines the non-Saccharomyces yeasts

T. delbrueckii and K. thermotolerans with S. cerevisiae (Anon.,
2004b). According to the technical data sheet this combination
(simultaneous inoculation) leads to wines with a richer and
rounder flavour with enhanced fruity notes. Improvements in
wine quality have been observed for a number of white
(Chardonnay, Pinot blanc, Pinot gris, Riesling) and red grape
varieties (Cabernet Sauvignon, Pinot noir, Shiraz, Merlot). The
proportion of the three different yeasts to each other is not dis-
closed, but the non-Saccharomyces to S. cerevisiae ratio appears
to be in the region of 1:14 (Jolly, unpublished data, 2005).
RESEARCH TRENDS IN RELATION TO NON-SACCHARO-
MYCES YEASTS
The utilisation of non-Saccharomyces yeasts such as C. stellata,
T. delbrueckii and others to improve the analytical profile and
flavour of wines has already been discussed. Concurrently with
the envisioned use of these new wine yeasts, new fermentation
techniques could be implemented and alternative alcoholic bever-
ages produced (Ciani & Maccarelli, 1998). It has already been
shown that non-Saccharomyces yeasts normally found during
wine fermentation are also responsible for the fermentation of
Kombucha, a traditional beverage produced by fermenting sweet-
ened black tea (Teoh et al., 2004). Non-Saccharomyces isolates
from wine could therefore be screened for improved strains for
Kombucha production.
It has also been shown that S. cerevisiae can initiate biofilm for-
mation (Reynolds & Fink, 2001), a characteristic previously
thought to be restricted to bacteria (Parsek & Greenberg, 2005).
This has implications for wine fermentation and the storage of
wines. The ability of wine-related non-Saccharomyces to form
biofilms is not well researched, but it has been suggested that yeast
cells on the surfaces of grape berries may interact in a biofilm sys-
tem (Renouf et al., 2005). It is also known that C. albicans, previ-
ously isolated from grapes (Parish & Caroll, 1985), forms biofilms
which contribute to its pathogenesis (Douglas, 2003). It can there-
fore be speculated that other non-Saccharomyces yeasts, even if
unable to initiate biofilms themselves, may become constituents of
a S. cerevisiae or C. albicans biofilm and, in so doing, contribute
positively or negatively to wine production.
As S. cerevisiae is glucophilic (a preference for glucose above
fructose) (Ough & Amerine, 1963; Margalith, 1981; Berthels et
al., 2004), the use of fructophilic non-Saccharomyces yeasts has
also been suggested for the remediation of some types of sluggish
or stuck fermentations (Gafner et al., 2000). Should the glucose
be utilised faster than the fructose during a normal fermentation,
then a glucose-fructose imbalance can occur and S. cerevisiae is
unable to ferment further. Under these circumstances fructophilic
yeasts, e.g. C. stellata and Z. bailii, which have a preference for
fructose, can be used to metabolise the fructose (Stterlin et al.,
2004). Once the glucose-fructose balance is restored, S. cerevisi-
ae should start fermenting again. An active dried experimental Z.
bailii strain has already been produced and is currently being
evaluated by commercial wine cellars (P. Loubser, Lallemand,
South Africa, personal communication, 2006). This technology
holds great potential as sluggish and stuck fermentations are a
common occurrence in all wine industries.
In another study, killer toxins from P. anomala and
Kluyveromyces wickerhamii have been investigated as antimicro-
bial agents against the spoilage yeast Dekkera/Brettanomyces
(Comitini et al., 2004). This has a potential application during
wine maturation and storage.
Apart from the investigations and experimentation with non-
Saccharomyces yeasts in wine production, other uses for non-
Saccharomyces yeasts and their metabolites are being sought.
These include the use of Pichia pastoris for the biomodification
of citrus aroma oil to improve and add value to the essence
(Goodrich et al., 1998). A protease enzyme isolated from
D. hansenii has also been suggested for hydrolysis of muscle pro-
teins during meat processing (Bolumar et al., 2005).
Another application for non-Saccharomyces yeasts is the use of
Zygocin, a protein toxin produced and secreted by killer strains of
Z. bailii (Weiler & Schmitt, 2003). Purified forms of the toxin
have potential as an antimycotic for a variety of human and phy-
topathogenic fungi (Schmitt & Breinig, 2002). The use of non-
Saccharomyces as bio-control alternatives to chemicals has also
been investigated. A sister species to M. pulcherrima (teleo-
morph of C. pulcherrima), Metschnikowia fructicola isolated
from grapes, shows activity against botrytis rot on stored grapes
(Kurtzman & Droby, 2001), while in vitro experiments show that
P. membranifaciens is antagonistic towards the causative agent,
Botrytis cinerea (Masih et al., 2001). Table grape storage and post
harvest damage due to botrytis is a major problem in grape-pro-
ducing regions that lie far from their respective domestic and
35
international markets. The possible use of Pichia guilliermondii
for copper uptake in bio-remediation of sewage sludge has also
been suggested (De Silniz et al., 2002). Considering the wide
biodiversity of yeasts found on grapes and in must, the potential
for finding yeasts benefiting mankind outside the parameters of
wine production is large.
FINAL COMMENTS
It is generally accepted that the wealth of yeast biodiversity with
hidden potential, especially for oenology, is largely untapped
(Pretorius, 2000). However, in order to exploit the potential ben-
efits of non-Saccharomyces yeasts in wine production and to min-
imise potential spoilage, the yeast populations on grapes and in
must, as well as the effect of wine making practices on these
yeasts, must be known, as must the metabolic characteristics of
non-Saccharomyces yeasts (Romano et al., 2003). This knowl-
edge will help realise the predictions of Heard (1999) concerning
the use of mixed starter cultures. His vision includes the use of
mixed yeast starter cultures tailored to reflect the characteristics
of a given wine region and the use of indigenous yeast species
with modern technology to produce novel wine-based beverages.
Strain selection will be very important, as not all strains within
a species will necessarily show the same desirable characteristics.
For example, significant variability is found in the formation of
undesirable biogenic amines amongst strains within some non-
Saccharomyces yeast species (Caruso et al., 2002).
Whatever the outcome of the search for non-Saccharomyces
yeasts for use in wine production, the accepted list of desirable
characteristics as pertaining to the wine yeast S. cerevisiae (Yap,
1987; Henschke, 1997; Pretorius, 2000) will not necessarily
apply to non-Saccharomyces yeasts. High fermentation efficien-
cy, high sulphite tolerance and zymocidal (killer) properties, for
example, might not be needed in the new technology of wine pro-
duction. The new non-Saccharomyces wine yeasts will necessar-
ily have a different list of desired characteristics. Furthermore, the
already mentioned problems envisioned by Erten & Campbell
(2001) regarding the need for non-standard fermentation equip-
ment and the reluctance of wine producers to cultivate and use, on
a large-scale, microorganisms generally considered as spoilage
organisms (Loureiro & Malfeito-Ferreira, 2003), should be noted.
Intensive education will have to accompany any new non-
Saccharomyces technology in wine production. However, the
goals as set out by Pretorius (2000; 2003) regarding efficient
sugar utilisation, enhanced production of desirable volatile esters,
enhanced liberation of grape terpenoids and production of glyc-
erol to improve wine flavour and other sensory properties can be
met by selected non-Saccharomyces wine yeasts. This path will
bypass current controversies regarding the genetic modification
of the workhorse of wine production, i.e. the wine yeast S. cere-
visiae. After the acceptance of genetically modified organisms
(GMO) by wine consumers and industries, genetic modification
of selected non-Saccharomyces yeasts can further enhance their
performance and role in wine production.
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HARMONY.nsac) combines the non-Saccharomyces yeasts

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